U.S. patent number 7,959,941 [Application Number 11/622,955] was granted by the patent office on 2011-06-14 for bone graft comprising a demineralized bone matrix and a stabilizing agent.
This patent grant is currently assigned to Warsaw Orthopedic, Inc.. Invention is credited to Michele Diegman, Nanette Forsyth, David Knaack, Kathy Traianedes, John Winterbottom.
United States Patent |
7,959,941 |
Knaack , et al. |
June 14, 2011 |
Bone graft comprising a demineralized bone matrix and a stabilizing
agent
Abstract
An improved demineralized bone matrix (DBM) or other matrix
composition is provided that has been mixed with a stabilizing
agent that acts as (1) a diffusion barrier, (2) a enzyme inhibitor,
(3) a competitive substrate, or (4) a masking moiety. A diffusion
barrier acts as a barrier so as to protect the osteoinductive
factors found in DBM from being degraded by proteolytic and
glycolytic enzymes at the implantation site. Stabilizing agents may
be any biodegradable material such as starches, modified starches,
cellulose, dextran, polymers, proteins, and collagen. As the
stabilizing agents degrades or dissolves in vivo, the
osteoinductive factors such as TGF-.beta., BMP, and IGF are
activated or exposed, and the activated factors work to recruit
cells from the preivascular space to the site of injury and to
cause differentiation into bone-forming cells. The invention also
provides methods of preparing, testing, and using the inventive
improved osteodinductive matrix compositions.
Inventors: |
Knaack; David (Summit, NJ),
Traianedes; Kathy (Malvern, AU), Diegman; Michele
(Scotch Plains, NJ), Forsyth; Nanette (Bayville, NJ),
Winterbottom; John (Jackson, NJ) |
Assignee: |
Warsaw Orthopedic, Inc.
(Warsaw, IN)
|
Family
ID: |
26986673 |
Appl.
No.: |
11/622,955 |
Filed: |
January 12, 2007 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20080145392 A1 |
Jun 19, 2008 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10271140 |
Oct 15, 2002 |
7163691 |
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60392462 |
Jun 27, 2002 |
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60329156 |
Oct 12, 2001 |
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Current U.S.
Class: |
424/426 |
Current CPC
Class: |
A61L
27/227 (20130101); A61L 27/3633 (20130101); A61L
27/58 (20130101); A61L 27/365 (20130101); A61F
2/28 (20130101); A61L 27/3608 (20130101); A61P
19/00 (20180101); A61L 27/54 (20130101); A61L
27/505 (20130101); A61L 2300/802 (20130101); A61L
2300/602 (20130101); A61F 2002/30677 (20130101); A61F
2002/2817 (20130101); A61L 2430/02 (20130101); A61L
2300/414 (20130101); B33Y 80/00 (20141201); A61L
2300/434 (20130101); A61F 2002/2835 (20130101) |
Current International
Class: |
A61F
2/28 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
179 833 |
|
Feb 1905 |
|
DE |
|
44 34 459 |
|
Apr 1996 |
|
DE |
|
29608321 |
|
Aug 1996 |
|
DE |
|
0 082 621 |
|
Jun 1983 |
|
EP |
|
0 243 151 |
|
Oct 1987 |
|
EP |
|
0 267 015 |
|
May 1988 |
|
EP |
|
0 321 442 |
|
Jun 1989 |
|
EP |
|
0 366 029 |
|
May 1990 |
|
EP |
|
0 406 856 |
|
Jan 1991 |
|
EP |
|
0405429 |
|
Jan 1991 |
|
EP |
|
0 411 925 |
|
Feb 1991 |
|
EP |
|
0 413 492 |
|
Feb 1991 |
|
EP |
|
0 419 275 |
|
Mar 1991 |
|
EP |
|
0 483 944 |
|
May 1992 |
|
EP |
|
0 495 284 |
|
Jul 1992 |
|
EP |
|
0 520 237 |
|
Dec 1992 |
|
EP |
|
0 555 807 |
|
Aug 1993 |
|
EP |
|
0567391 |
|
Oct 1993 |
|
EP |
|
0 693 523 |
|
Jan 1996 |
|
EP |
|
1 142 581 |
|
Oct 2001 |
|
EP |
|
2691901 |
|
Dec 1993 |
|
FR |
|
2175807 |
|
Oct 1986 |
|
GB |
|
9059/1986 |
|
Mar 1986 |
|
JP |
|
2121652 |
|
May 1990 |
|
JP |
|
3210270 |
|
Sep 1991 |
|
JP |
|
4097747 |
|
Feb 1992 |
|
JP |
|
9506281 |
|
Jun 1997 |
|
JP |
|
0880425 |
|
Nov 1981 |
|
SU |
|
WO 86/07265 |
|
Dec 1986 |
|
WO |
|
WO 89/04646 |
|
Jun 1989 |
|
WO |
|
89/11880 |
|
Dec 1989 |
|
WO |
|
WO 94/21196 |
|
Sep 1994 |
|
WO |
|
95/15776 |
|
Jun 1995 |
|
WO |
|
96/39203 |
|
Dec 1996 |
|
WO |
|
WO 97/25941 |
|
Jul 1997 |
|
WO |
|
WO 98/00183 |
|
Jan 1998 |
|
WO |
|
WO 98/40113 |
|
Sep 1998 |
|
WO |
|
WO 99/39757 |
|
Aug 1999 |
|
WO |
|
WO 00/34556 |
|
Jun 2000 |
|
WO |
|
WO 00/35510 |
|
Jun 2000 |
|
WO |
|
WO 00/50102 |
|
Aug 2000 |
|
WO |
|
WO 01/08584 |
|
Aug 2001 |
|
WO |
|
WO 02/02156 |
|
Jan 2002 |
|
WO |
|
WO 02/47587 |
|
Jun 2002 |
|
WO |
|
2004/108023 |
|
Dec 2004 |
|
WO |
|
WO 2006/057011 |
|
Jun 2006 |
|
WO |
|
WO 2006/076712 |
|
Jul 2006 |
|
WO |
|
Other References
Crowe et al., "Inhibition of Enzymatic Digestion of Amylose by Free
Fatty Acids in Vitro Contributes to Resistant Starch Formation", J.
Nutr. 130(8): 2006-2008, 2000. cited by other .
Edwards et al., "Osteoinduction of Human Demineralized Bone:
Characterization in a Rat Model", Clinical Orthopeadics & Rel.
Res., 357:219-228, 1998. cited by other .
Glowacki et al., "Demineralized Bone Implants", Clinics in Plastic
Surgery, 12(2): 233-241,1985. cited by other .
Kubler, et al., "Erstes BMP-Analog Mit Osteoinduktiven
Eigenschaften", Mund Kiefer Gesichtschir, 3 Suppl. 1: S134-S139,
1999. cited by other .
Kubler et al., "Allogenic Bone and Cartilage Morphogenesis", J.
Craniomaxillofac. Surg. 19(7): 238-288, 1991. cited by other .
Mulliken et al., "Fate of Mineralized and Demineralized Osseous
Implants in Cranial Defects", Calcified Tissue Int. 33: 71-76,
1981. cited by other .
Neigal et al., "Use of Demineralized Bone Implants in Orbital and
Craniofacial", Opthal. Plast. Recontrs. Surg., 12: 108-120, 1996.
cited by other .
Ray et al., "Preliminary Report of an Experimental Study", J. Bone
Joint Surgery, 39 A:1119-1128, 1957. cited by other .
Russell et al., "Clinical Utility of Demineralized Bone Matrix for
Osseous Defects, Arthrodesis and Reconstruction: Impact of
Processing Techniques and Study Methodology", Orthopaedics, 22(5):
524-531, 1999. cited by other .
Ueland et al., "Increased Cortical Bone Content of Insulin-Like
Growth Factors in Acromegalic Patients", J. Clin. Endocrinol.
Metab., 84(1): 123-127, 1999. cited by other .
Urist, "Bone: Formation by Autoinduction", Science, 150: 893-899,
1965. cited by other .
Whiteman et al., "Demineralized Bone Powder--Clinical Applications
for Bone Defects of the Hand", J. Hand. Surg., 18B: 487-490, 1993.
cited by other .
Whittaker et al., "Matrix Metalloproteinases and Their
Inhibitors--Current Status and Future Challenges",
Celltransmissions, 17(1):3-14. cited by other .
Xiabo et al., "Experimental and Clinical Investigations of Human
Insoluble Bone Matrix Gelatin", Clin. Orthrop. 293:360-365, 1993.
cited by other .
Zhang, et al., "A Quantitative Assessment of Osteoinductivity of
Human Demineralized Bone Matrix", J. Periodontol. 68(11):
1076-1084, 1997. cited by other .
Stairs, Robert A. "Calculation of surface tension of salt
solutions: effective polarizability of solvated ions." Can. J.
Chem. 73: pp. 781-787 (1995). cited by other .
Abel, E. "The vapor phase above the system sulfuric acid-water." J.
Phys. Chem. 50(3), pp. 260-283 (1946). cited by other .
Abjornson et al., "A Novel Approach to Bone Grafting Substitutes",
Society for Biomaterials, p. 1372 (2000). cited by other .
Block, Michael S., D.M.D. et al., "Bone Maintenance 5 to 10 years
After Sinus Grafting", J. Oral Maxillofacial Surg., vol. 56, pp.
706-714, 1998. cited by other .
Bobyn et al., "The Optimum Pore Size for the Fixation of
Porous-Surfaced Metal Implants by Ingrowth of Bone", Clinical
Orthopaedics and Related Research, 1980, pp. 263-270. cited by
other .
Bolander et al.," The Use of Demineralized Bone Matrix ion te
Repair of Segmental Defects", The Journal of Bone and Joint
Surgery, vol. 68-A, No. 8, pp. 1264-1273. cited by other .
Bostrom et al., "Use of Bone Morphogeneic Protein-2 in the Rabbit
Ulnar Nonunion Model", Clinical Orthopaedics and Related Research,
No. 327, pp. 272-282 (1996). cited by other .
Covey et al., "Clinical Induction of Bone Repair with Demineralized
Bone Matrix or a Bone Morphogenetic Protein", Orthopaedic Review,
Aug. 1989, vol. XVIII, No. 8, pp. 857-863. cited by other .
Gekko et al., "Mechanism of Protein Stabilization by Glycerol:
Preferential Hydration in Glycerol-Water Mixtures", vol. 20, No.
16, pp. 4667-5676 (1981). cited by other .
Gepstein et al., "Bridging Large Defects in Bone by Demineralized
Bone Matrix in the Form of a Powder", The Journal of Bone and Joint
Surgery, vol. 69-A, No. 7, pp. 984-991, 1987. cited by other .
Gher, Marlin E., et al., "Bone Grafting and Guided Bone
Regeneration for Immediate Dental Implants in Humans", J.
Periodontology, 1994, 65:881-891. cited by other .
Glowacki et al., "Application of Biological Principle of Induced
Osteogenesis for Craniofacial Defects", The Lancet, 1981, vol. 1,
No. 8227, pp. 959-962. cited by other .
Groeneveld et al., "Mineralized Processes in Demineralized Bone
Matrix Grafts in Human Maxillary Sinus Floor Elevations", John
Wiley & Sons, Inc. pp. 393-402 (1999). cited by other .
Habal et al., "Autologous Corticocancellous Bone Paste for Long
Bone Discontinuity Defects: An Experimental Approach", Annals of
Plastic Surgery, Aug. 1985, vol. 15, No. 23, pp. 138-142. cited by
other .
Ito, Takayasu et al., "Sensitivity of Osteoinductive Activity of
Demineralized and Defatted Rat Femur to Temperature and Furation of
Heating", Clinical Orthopaedics and Related Research, No. 316,
1995, pp. 267-275. cited by other .
Jurgensen, K., M.D. et al., "A New Biological Glue for
Cartilage-Cartilage-Cartilage Interfaces: Tissue Transglutaminase",
Journal of Bone and Joint Surgery, Inc., Feb. 1997, pp. 185-193.
cited by other .
Kaban et al., "treatment of Jaw Defects with Demineralized Bone
Implants", Journal of Oral and Maxillofacial Surgery, pp. 623-626
(Jun. 6, 1998). cited by other .
Kakiuchi et al., "Human Bone Matrix Gelatin as a Clinical
Alloimplant", International Orthopaedics, 9, pp. 181-188 (1985).
cited by other .
Kiviranta et al., "The Rate fo Calcium Extraction During EDTA
Decalcification from Thin Bone Slices as Assessed with Atomic
Absorption Spectrophometry", Histochemistry 68, 1980, pp. 119-127.
cited by other .
Lewandrowski et al., "Flexural Rigidity in Partially Demineralized
Diaphysical Bone Grafts," Clin. Ortho. Rel. Res. 317: 254-262,
1995. cited by other .
Lewandrowski et al., "Kinetics of Cortical Bone Demineralization:
controlled demineralization--a new method for modifying cortical
bone allografts," J. Biomed. Mater. Res. 31:365-372, 1996. cited by
other .
McLaughlin et al., "Enhancements of Bone Ingrowth by the Use of
Bone Matrix as a Biologic Cement", Clinical Orthopaedics and
Related Research, No. 183, pp. 255-261 (Mar. 1984). cited by other
.
Meijer et al., Radiographic Evaluation of Mandibular Augmentation
with Prefabricated Hydroxylapatite/Fibrin Glue Imlants, Journal of
Oral and Maxillofacial Surgery, 1997, pp. 138-145. cited by other
.
Mellonig, "Decalicified Freeze-Dried Bone Allograft as an Implant
Material in Human Periodontal Defects", The International Journal
of Periodontics and Restorative Dentistry, pp. 41-45, 1984. cited
by other .
Mellonig, James T. D.D.S., M.S., "Bone Allografts in Periodontal
Therapy", Clinical Orthopaedics and Related Research, No. 324, Mar.
1996. cited by other .
Mulliken, J.B. and Glowacki, "Induced Osteogenesis for Repair and
Construction in the Craniofacial Region", J. Plastic and
Reconstructive Surgery, May 1980, p. 553-559. cited by other .
Paralkar, et al., PNAS, 100(11): 6736-6740, 2003. cited by other
.
Parma-Benfenati, S., et al., "Histologic Evaluation of New
Attachment Utilizing a Titanium-Reinforced Barrier Membrane in a
Nucogingival Recession Defect. A Case Report", J. Periodontology,
Jul. 1998. cited by other .
Perez, B.J. et al., "Mechanical properties of a discontinous random
fiber composite for totally bioabsorbable fracture fixation
devices", Paper presented in : Bioengineering Conference, 1995,
Proceedings of the 1995 IEEE 21st Annual Northeast, May 22-23,
1995, pp. 55-56. cited by other .
Product literature for Bio-Gide.RTM., Resorbable barrier membrane
from OsteoHealth Co., Division of Luitpold Pharmaceutical, Inc.
1998. cited by other .
Product literature for Gore Resolut XT, Bioabsorbable membrane from
Gore Regenerative Technologies, Palm Beach Gardens, FL 1998. cited
by other .
Reddi et al., Proc. Natl. Acad. Sci. 69:1601-1605, 1972. cited by
other .
Stevenson et al., "Factors Affecting Bone Graft Incorporation",
Clinical Orthopaedics and Related Research, No. 323, 1996, pp.
66-74. cited by other .
The Term "Substantially", Merriam-Webster Online Dictionary, at the
web--http://www.m-w.com, p. 1. cited by other .
Teparat, Thitiwan et al., "Clinical Comparison of Bioabsorbable
Barriers With Non-Resorbable Barriers in Guided Tissue Regeneration
in the Treatment of Human Intrabony Defects", J. Periodontology,
Jun. 1998. cited by other .
Todescan et al., "A Small Animal Model for Investigating Endosseous
Dental Impants:Effect of Graft Materials on Healing of Endoss,
Porous-Surfaced Implants Placed in a Fresh Extraction Socket", The
Journal of Oral and Maxillofacial Implants, vol. 2, No. 2, pp.
217-223, 1987. cited by other .
Urist, M.R. et al., "The Bone Induction Principle", Clin. Orthop.
Rel. Res. 53:243-283, 1967. cited by other .
Ruppert, Rainer et al. "Human bone morphogenetic protein 2 contains
a heparin-binding site which modifies its biological activity,"
Eur. J. Biochem, 237(1): 295-302 (1996). cited by other .
Kubler, N. R. et al. "EHBMP-2: The first BMP-variant with
osteoinductive properties," Mund Kiefer Gesichtschir, 3(1):
S134-S139 (1999). cited by other .
Reddi, A. Hari. "Interplay between bone morphogenetic proteins and
cognate binding proteins in bone and cartilage development: noggin,
chordin and DAN," Arthritis Research, 3(1): 1-5 (2001). cited by
other .
Gazzerro, Elisabetta et al. "Bone Morphogenetic Proteins Induce the
Expression of Noggin, Which Limits Their Activity in Cultured Rat
Osteoblasts," Jour. of Clin. Invest., 102(12): 2106-2114 (1998).
cited by other .
Yamaguchi, Akira. "Recent advances in researches on bone
formation--Role of BMP in bone formation," Nihon Rinsyo, 56(6):
1406-1411 (1998). cited by other .
Dallas, Sarah L. et al. "Dual Role for the Latent Transforming
Growth Factor-.beta. Binding Protein in Storage of Latent
TGF-.beta. in the Extracellular Matrix and as a Structural Matrix
Protein," Jour. of Cell Biol., 131(2): 539-549 (1995). cited by
other .
Pedrozo, Hugo A. et al. "Vitamin D.sub.3 Metabolites Regulate LTBP1
and Latent TGF-.beta.1 Expression and Latent TGF.beta.1
Incorporation in the Extracellular Matrix of Chohdrocytes," Jour.
of Cell. Biochem., 72(1): 151-165 (1999). cited by other .
Pedrozo, Hugo A. et al. "Growth Plate Chondrocytes Store Latent
Transforming Growth Factor (TGF)-.beta.1 in Their Matrix Through
Latent TGF-.beta.1 Binding Protein-1," Jour. of Cellular
Physiology, 177(2): 343-354 (1997). cited by other .
Bautista, Catalino M. et al. "Isolation of a novel insulin-like
growth factor (IGF) binding protein from human bone: A potential
candidate for fixing IGF-II in human bone," Biochem. and Biophys.
Research Communications, 176(2): 756-763 (Apr. 30, 1991). cited by
other .
Mohan, S. "Insulin-Like Growth Factor Binding Proteins in Bone Cell
Regulation," Growth Regulation, 3(1): 67-70 (1993). cited by other
.
Japanese Office Action dated Mar. 18, 2009, from related,
co-pending application JP 2003-533987. cited by other .
Jada, vol. 133, Dec. 2002.
http://jada.ada.org/cgi/reprint/133/12/1610-a. cited by other .
Urist et al. "Bone Formation in Implants of Partially and Wholly
Demineralized Bone Matrix," Clinical Orthopaedics and Related
Research, vol. 71, pp. 271-278 (1970). cited by other .
Grafton.TM. Allogenic Bone Matrix (ABM), Advertising Brochure,
Advanced Processing of Human Allograft Bone, Osteotech, Inc., 1992.
cited by other .
Frenkel et al. "Use of Demineralized Bone Matrix Gel to Enhance
Spine Fusion", 19.sup.th Annual Meeting of the Society for
Biomaterials, Apr. 28-May 2, 1993, Birmingham, AL, p. 162. cited by
other .
Stevenson et al. "Long Bone Defect Healing Induced by a New
Formulation of Rat Demineralized Bone Matrix Gel," 40.sup.th Annual
Meeting, Orthopedic Research Society, Feb. 21-24, 1994, New
Orleans, LA, p. 205-35. cited by other.
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Primary Examiner: Azpuru; Carlos A
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a divisional application of U.S. patent application Ser.
No. 10/271,140, filed on Oct. 15, 2002, now U.S. Pat. No.
7,163,691, which claims priority to U.S. Application Ser. No.
60/392,462, filed Jun. 27, 2002, and U.S. Application Ser. No.
60/329,156, filed Oct. 12, 2001, the contents of which are
incorporated herein by reference in its entirety.
Claims
The invention claimed is:
1. An implantable bone growth composition comprising: demineralized
bone matrix; and a stabilizing agent comprising a masking entity
chosen from the group consisting of a growth factor binding protein
and a lectin.
2. The composition of claim 1, wherein the growth factor binding
protein is selected from the group consisting of noggin, chordin,
follistatin, a TGF-.beta. binding protein, and an insulin-like
growth factor.
3. The composition of claim 1, further comprising an agent that
induces release of a growth factor.
4. The composition of claim 3, wherein the agent is encapsulated in
a biodegradable polymer such that the growth factor is released
over an extended period of time.
5. The composition of claim 1, further comprising a competitive
substrate.
6. The composition of claim 5 wherein the competitive substrate
comprises a polylysine.
7. The composition of claim 5, wherein the competitive substrate
comprises a polysaccharide.
8. The composition of claim 1, further comprising an enzyme
inhibitor.
9. The composition of claim 8, wherein the enzyme inhibitor
comprises a protease inhibitor.
10. The composition of claim 1, further comprising a diffusion
barrier.
11. The composition of claim 10, wherein the diffusion barrier
comprises a resorbable polymer.
12. The composition of claim 10, wherein the diffusion barrier
comprises an enzymatically degraded polymer.
13. The composition of claim 10, wherein the diffusion barrier
comprises a fatty acid.
14. The composition of claim 10, wherein the diffusion barrier
comprises a phospholipid.
15. The composition of claim 10, wherein the diffusion barrier
comprises a polysaccharide.
16. The composition of claim 15, wherein the polysaccharide is
selected from the group of starches and celluloses.
17. The implant of claim 1, wherein the composition exhibits an
osteoinductivity that is at least about 10% greater than that of a
composition of demineralized bone matrix without the stabilizing
agent.
18. The composition of claim 1, wherein the composition exhibits an
osteoinductivity that is at least about 20% greater than that of a
composition of demineralized bone matrix without the stabilizing
agent.
19. The composition of claim 1, wherein the composition exhibits an
osteoinductivity that is at least about 35% greater than
osteoinductivity of a composition of demineralized bone matrix
without the stabilizing agent.
20. The composition of claim 1, wherein the demineralized bone
matrix comprises particles that are tapered, wedge-shaped, or
cone-shaped; and wherein the particles are at least about 1 mm in
their largest dimension and wherein the particles are about 100
microns in another dimension.
21. The composition of claim 1, wherein the composition exhibits an
osteoinductivity of at least about 25% of an osteoinductivity of a
10 .mu.g BMP-collagen based sponge preparation.
22. The composition of claim 1, wherein the composition further
comprises a drug to be delivered.
23. The composition of claim 22, wherein the drug to be delivered
is an osteoinductive growth factor.
Description
BACKGROUND
The rapid and effective repair of bone defects caused by injury,
disease, wounds, surgery, etc., has long been a goal of orthopaedic
surgery. Toward this end, a number of compositions and materials
have been used or proposed for use in the repair of bone defects.
The biological, physical, and mechanical properties of the
compositions and materials are among the major factors influencing
their suitability and performance in various orthopaedic
applications.
Autologous cancellous bone ("ACB") is considered the gold standard
for bone grafts. ACB is osteoinductive, is non-immunogenic and, by
definition, has all of the appropriate structural and functional
characteristics appropriate for the particular recipient.
Unfortunately, ACB is only available in a limited number of
circumstances. Some individuals lack ACB of appropriate dimensions
and quality for transplantation. Moreover, donor site morbidity can
pose serious problems for patients and their physicians.
Much effort has been invested in the identification or development
of alternative bone graft materials. Demineralized bone matrix
("DBM") implants have been reported to be particularly useful (see,
for example, U.S. Pat. Nos. 4,394,370; 4,440,750; 4,485,097;
4,678,470; and 4,743,259; Mulliken et al., Calcif. Tissue Int.
33:71, 1981; Neigel et al., Opthal. Plast. Reconstr. Surg. 12:108,
1996; Whiteman et al., J. Hand. Surg. 18B:487, 1993; Xiaobo et al.,
Clin. Orthop. 293:360, 1993; each of which is incorporated herein
by reference). Demineralized bone matrix is typically derived from
cadavers. The bone is removed aseptically and/or treated to kill
any infectious agents. The bone is then particulated by milling or
grinding and then the mineral component is extracted (e.g., by
soaking the bone in an acidic solution). The remaining matrix is
malleable and can be further processed and/or formed and shaped for
implantation into a particular site in the recipient. Demineralized
bone prepared in this manner contains a variety of components
including proteins, glycoproteins, growth factors, and
proteoglycans. Following implantation, the presence of DBM induces
cellular recruitment to the site of injury. The recruited cells may
eventually differentiate into bone forming cells. Such recruitment
of cells leads to an increase in the rate of wound healing and,
therefore, to faster recovery for the patient.
Current DBM formulations have various drawbacks. First, while the
collagen-based matrix of DBM is relatively stable, the active
factors within the DBM matrix are rapidly degraded. The osteogenic
activity of the DBM may be significantly degraded within 24 hours
after implantation, and in some instances the osteogenic activity
may be inactivated within 6 hours. Therefore, the factors
associated with the DBM are only available to recruit cells to the
site of injury for a short time after transplantation. For much of
the healing process, which may take weeks to months, the implanted
material may provide little or no assistance in recruiting
cells.
In addition to the active factors present within the DBM, the
overall structure of the DBM implant is also believed to contribute
to the bone healing capabilities of the implant.
SUMMARY OF THE INVENTION
The present invention provides improved demineralized bone matrix
("DBM") compositions, related methods for preparing and using the
inventive compositions, and kits containing the inventive
compositions. The invention encompasses the recognition that the
fast reduction in osteoinductive capabilities observed with
previously available DBM compositions may result from (1)
degradation of osteoinductive agents, for example, as a result of
proteases, sugar-degrading enzymes, or other enzymes present in the
host or the DBM itself, (2) diffusion of osteoinductive agents out
of the DBM; and/or (3) reduced activation of osteoinductive agents
in the DBM. The present invention therefore provides DBM
compositions in which osteoinductive agents are protected from
degradation and/or from diffusion out of the composition. The
present invention may also include activation of the osteoinductive
factors found in the DBM, for example, in a controlled time release
manner. In some embodiments, the invention also provides improved
shape-retaining characteristics contributing to the overall
efficacy of the DBM compositions. Also, in some embodiments, the
inventive DBM composition can be used as a delivery device to
administer bioactive agents.
Protection of the active factors within the DBM is provided using
(1) diffusion barriers (e.g., polymers, starch), (2) enzyme
inhibitors (e.g., protease inhibitors), (3) competitive substrates,
and/or (4) masking moieties. Certain embodiments of the invention
provide DBM compositions comprising a stabilizing agent such as a
polymer or other factor (e.g., protease inhibitors). Preferably,
the polymer as a diffusion barrier is metabolized over time, so
that the osteoinductive agents are unmasked and/or released from
the DBM composition over time, or retarded in their degradation
rate. Diffusion barriers of the invention may also work through
alternative means by decreasing the diffusion of the activating
enzymes to the factors present in the DBM composition. Preferably,
such unmasking, release, controlled release, or controlled
degradation occurs over a period longer than several hours,
preferably longer than a day to several days, and possibly lasting
weeks or even months. In certain preferred embodiments, the rates
of degradation, release, and activation are balanced to yield a DBM
composition with the desired level of osteoinductivity over time.
Inventive compositions containing a stabilizing agent typically
show osteoinductive activity for longer periods of time than is
seen with comparable compositions lacking the stabilizing
agent.
In some embodiments of the invention, the stabilizing agent may
comprise a polymer, such as a biodegradable polymer (e.g., that
inhibits or delays diffusion of osteoinductive agents out of the
DBM composition, and/or blocks access of degrading and/or
activating enzymes to the osteoinductive agents). Examples of
biodegradable polymers include starches, dextrans, cellulose,
poly-esters, proteins, polycarbonates, polyarylates, and PLGA.
Preferably the polymers are biocompatible and biodegradable.
In other embodiments, inventive DBM compositions may include and/or
be treated with agents that inhibit the activity of one or more
activating enzymes, proteases, or glycosidases. Such inhibitory
agents are expected to reduce the activity of specific enzymes
(whether derived from the host or from the DBM) that would
otherwise interact with osteoinductive agents or other active
agents in the DBM, thereby reducing osteoinductivity or wound
healing. Alternatively or additionally, inventive DBM compositions
may include inhibitory agents presented in a time-release
formulation (e.g., encapsulated in a biodegradable polymer). In the
case of activating enzymes (i.e., enzymes which lead to the
release, presentation, or creation of osteoinductive factors),
inhibitory agents that reduce the activity of activating enzymes
preferably lead to increased osteoinductivity over an extended
period of time rather than just a burst just after
implantation.
Some embodiments of the present invention comprise DBM compositions
particularly formulated to control or adjust the rate by which the
composition, or portions thereof, lose osteoinductivity. To give
but one example, DBM compositions may be prepared from multiple
different DBM preparations, each of which contains DBM particles of
different size and/or including different amounts or types of
stabilizing agents. For instance, DBM preparations or powders may
be prepared that have varying half-lives as determined by changing,
for instance, the nature or amount of a stabilizing polymer, the
extent of cross-linking of the polymer, the thickness of a
stabilizing coating, the size of the particles, the amount of
inhibitors of activating or degradatory enzymes, etc. Adjusting the
amounts or locations of the different DBM preparations within the
overall inventive DBM composition can modify the characteristics of
part or all of the inventive composition. In this manner, for
example, the formulation could be customized to the patient, type
of injury, site of injury, length of recovery, underlying disease,
etc.
In another aspect, the present invention provides methods of
preparing inventive improved DBM compositions. For instance, the
present invention provides methods of formulating an improved DBM
composition for a particular site or injury.
The present invention also provides systems and reagents for
preparing and applying DBM grafts, as well as systems and reagents
for treating bone defects using DBM implants. For example, the DBM
composition may be provided as a paste in a delivery device such as
a syringe. Preferably, the DBM composition is sterile and is
packaged so that it can be applied under sterile conditions (e.g.,
in an operating room).
The present invention further provides a system for characterizing
DBM composites, and for identifying and preparing DBM-containing
materials with improved properties.
Furthermore, the present invention provides a system for delivering
bioactive agents, such as growth factors (e.g., bone morphogenic
proteins, growth factors, hormones, angiogenic factors, cytokines,
interleukins, osteopontin, osteonectin), to a host animal. The use
of a DBM composition as a delivery vehicle for bioactive agents
provides for the unexpected result of an improved healing response
to the implant without the need to administer separately the
bioactive agent. A problem with the introduction of the bioactive
agent at the site is that it is often diluted and redistributed
during the healing process by the circulatory systems (e.g., blood,
lymph) of the recipient before complete healing has occurred. A
solution to this problem of redistribution is to affix the
bioactive components to the osteoimplant. Some preferred bioactive
agents that can be delivered using a DBM composition include agents
that promote the natural healing process, i.e., resorption,
vascularization, angiogenesis, new growth, etc. A list of
biological agents that may be delivered using inventive DBM
compositions is included as Appendix A. In preferred embodiments of
this aspect of the invention, an inventive composition is provided
in which DBM, together with a stabilizing agent, is used to deliver
the biologically active agent. It is expected that the stabilizing
agent will protect the biologically active agent from degradation,
and therefore will extend its active life after delivery into the
recipient animal. In certain embodiments, the bioactive agent is an
osteoinductive agent, and in certain embodiments, the DBM may be
used to deliver more than one bioactive agent, preferably more than
two, and more preferably sometimes more than three bioactive
agents. The bioactive agent may be associated with the DBM. For
example, the bioactive agent may be associated with the DBM through
electrostatic interactions, hydrogen bonding, pi stacking,
hydrophobic interactions, van der Waals interactions, etc. In
certain embodiments, the bioactive agent is attached to the DBM
through specific interactions such as those between a receptor and
its ligand or between an antibody and its antigen. In other
embodiments, the bioactive agent is attached to the DBM through
non-specific interactions (e.g., hydrophobic interactions).
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1. Three week in-vivo radiographs showing evidence of bone
formation.
FIG. 2. Six week x-rays or faxitron images.
FIG. 3. Qualitative evaluation of Vascularity (A) and Residual
Demineralized Bone Fiber (DBF) (B).
A. The vascularity and marrow cellularity increased on active DBF
in a dose-dependent fashion with increasing concentrations of
hrhBMP-2.times., which was not evident in the devitalized group.
The wild type rhBMP-2 at the 5 .mu.g dose was similar to the hybrid
BMP.
B. The residual DBF remained a significant part of the nodule in
each of the devitalized groups. The residual DBF dose-dependently
deceased with increasing amounts of hrhBMP-2.times. in the active
DBF group. The wild type rhBMP-2 was not as effective in remodeling
the DBF as the hrhBMP-2.times.
FIG. 4. Comparison of untreated and hrhBMP-2.times. treated
devitalized and active DBF matrix.
Devitalized: Only residual DBF present with no bone formation
elements evident.
Devitalized+10 .mu.g hrhBMP-2.times.: New bone lining residual
bone; extensive immature marrow with many adipocytes throughout
nodule; extensive bone formation at outer edge of nodule but no rim
present.
DBF: Rim of residual DBF present with extensive chondrocytes, bone,
and some marrow formation.
DBF+10 .mu.g hrhBMP-2.times.: Thin rim of mature new bone with
extensive bone formation through out nodule with very little
residual DBF remaining at center; extensively vascularized with
well developed hematopoietic marrow present.
FIG. 5. Histological comparison of hrhBMP-2.times. and wild type
rhBMP-2 treated DBF matrix. There was significant bone formation in
the hrhBMP-2.times. treated group compared to the rhBMP-2 group as
evidenced by fewer spicules of bone and an extensive fatty marrow
in the wild type group. A more developed, blood marrow was evident
in the hybrid rhBMP-2.times. group.
FIG. 6. Chemical structure of some examples of matrix
metalloproteinase inhibitors.
DEFINITIONS
Associated with: A stabilizing agent or other chemical entity is
associated with DBM or other osteogenic matrix according to the
present invention if it is retained by the implant long enough to
significantly enhance the osteoinductivity of the implant. Specific
examples include 1) not freely diffusible from the DBM as
determined in in vitro diffusion assays in simulated body fluids;
and/or 2) has an extended half-life in the DBM as compared with
free in solution. In some embodiments, associations are covalent;
in others they are non-covalent. Examples of non-covalent
interactions include electrostatic interactions, hydrogen bonding,
hydrophobic interactions, and van der Waals interactions. For
instance, a bioactive agent may be rendered associated with a DBM
or other inventive matrix by virtue of a polymeric stabilizing
agent that restrains diffusion of the bioactive agent from the
matrix. Alternatively or additionally, the bioactive agent may be
rendered associated with a DBM by virtue of a physical interaction
with one or more entities that are themselves associated with the
DBM. For example, the BMP-2 in Example 12 is considered to be
associated with the DBM, and the BMP-2.times. is considered to be
more closely associated with the DBM than the BMP-2.
Demineralized bone activity refers to the osteoinductive activity
of demineralized bone.
Demineralized bone matrix, as used herein, refers to any material
generated by removing mineral material from living bone tissue. In
preferred embodiments, the DBM compositions as used herein include
preparations containing less than 5% calcium and preferably less
than 1% calcium by weight. Partially demineralized bone (e.g.,
preparations with greater than 5% calcium by weight but containing
less than 100% of the original starting amount of calcium) are also
considered within the scope of the invention.
Diffusion barrier refers to any material, coating, film, or
substance that decreases the rate of diffusion of a substance from
one side of the barrier to the other side, and more specifically,
from outside to in or vice versa. The diffusion barrier in certain
embodiments may be a polymer including proteins, polysaccharides,
cellulose, man-made polymer, PLGA, etc. that prevents the diffusion
of activating agents (including water, enzymes, etc.) and/or
degradatory enzymes into the DBM composition. The diffusion barrier
may also prevent the movement of osteoinductive factors out of the
DBM composition. In certain embodiments, the diffusion barrier is
biodegradable leading to the degradation, activation, or release of
osteoinductive factors over an extended period of time.
Matrix, as used herein, refers to a natural or non-natural
substantially solid vehicle capable of association with at least
one growth factor for delivery to an implant site. The matrix may
be completely insoluble or may be slowly solubilized after
implantation. Following implantation, preferred matrices resorb or
degrade, remaining substantially intact for at least one to seven
days, most preferably for two or four weeks or longer and often
longer than 60 days. Growth factors may be endogenously present on
the matrix as in the case of most demineralized bone, or they may
be exogenously added to the matrix. Matrices may also comprise
combinations of endogenous and exogenous growth factors. The matrix
may be in particulate or fiber form, or may be monolithic. The
matrix may comprise a number of materials and forms in combination
such as fibers and particles. In one preferred embodiment, the
matrix is comprised of heat pressed demineralized bone fibers. In
other embodiments, the matrix comprises resorbable plastic polymers
such as those described below as suitable for use as diffusion
barriers. In other preferred embodiments, a particulated amorphous
calcium phosphate is used as the matrix in association with an
adsorbed growth factor such as a BMP. More specifically BMP-2 or
BMP-4 or derivatives thereof. Still other matrix embodiments
requiring the addition of an exogenous growth factor include, but
are not limited to, particulated ceramics, preferably calcium
sulphates or calcium phosphates. The most preferred matrices are
calcium phosphates, the preparation of which is well known to
practitioners in the art (see, for example, Driessens et al.
"Calcium phosphate bone cements" Wise, D. L., Ed. Encyclopedic
Handbook of Biomaterials and Bioengineering, Part B, Applications
New York: Marcel Decker; Elliott Structure and Chemistry of the
Apatites and Other Calcium Phosphates Elsevier, Amsterdam, 1994;
each of which is incorporated herein by reference). Calcium
phosphate matrices include, but are not limited to, dicalcium
phosphate dihydrate, monetite, tricalcium phosphate, tetracalcium
phosphate, hydroxyapatite, nanocrystalline hydroxyapatite, poorly
crystalline hydroxyapatite, substituted hydroxyapatite, and calcium
deficient hydroxyapatites.
Osteoinductive, as used herein, refers to the quality of being able
to stimulate bone formation. Any material that can induce the
formation of ectopic bone in the soft tissue of an animal is
considered osteoinductive. For example, most osteoinductive
materials induce bone formation in athymic rats when assayed
according to the method of Edwards et al. ("Osteoinduction of Human
Demineralized Bone: Characterization in a Rat Model" Clinical
Orthopeadics & Rel. Res., 357:219-228, December 1998;
incorporated herein by reference). Osteoinductivity in some
instances is considered to occur through cellular recruitment and
induction of the recruited cells to an osteogenic phenotype.
Osteoinductivity may also be determined in tissue culture as the
ability to induce an osteogenic phenotype in culture cells
(primary, secondary, or explants) It is advisable to calibrate the
tissue culture method with an in vivo ectopic bone formation assay
as described by Zhang et al. "A quantitative assessment of
osteoinductivity of human demineralized bone matrix" J.
Periodontol. 68(11): 1076-84, November 1997; incorporated herein by
reference. Calibration of the in vitro assays against a proven in
vivo ectopic bone formation model is critical because the ability
of a compound to induce an apparent "osteogenic" phenotype in
tissue culture may not always be correlated with the induction of
new bone formation in vivo. BMP, IGF, TGF-.beta., parathyroid
hormone (PTH), and angiogenic factors are only some of the
osteoinductive factors found to recruit cells from the marrow or
perivascular space to the site of injury and then cause the
differentiation of these recruited cells down a line responsible
for bone formation. DBM isolated from either bone or dentin have
both been found to be osteoinductive materials (Ray et al., "Bone
implants" J. Bone Joint Surgery 39A:1119, 1957; Urist, "Bone:
formation by autoinduction" Science 150:893, 1965; each of which is
incorporated herein by reference).
Osteoinductivity score refers to a score ranging from 0 to 4 as
determined according to the method of Edwards et al. (1998) or an
equivalent calibrated test. In the method of Edwards et al., a
score of "0.infin. represents no new bone formation; "1" represents
1%-25% of implant involved in new bone formation; "2" represents
26-50% of implant involved in new bone formation; "3" represents
51%-75% of implant involved in new bone formation; and "4"
represents >75% of implant involved in new bone formation. In
most instances, the score is assessed 28 days after implantation.
However, for the improved inventive formulations, particularly
those with osteoinductivity comparable to the BMPs, the
osteoinductive score may be obtained at earlier time points such as
7, 14, or 21 days following implantation. In these instances it is
important to include a normal DBM control such as DBM powder
without a carrier, and if possible, a positive control such as BMP.
Occasionally osteoinductivity may also be scored at later
timepoints such as 40, 60, or even 100 days following implantation.
Percentage of osteoinductivity refers to an osteoinductivity score
at a given time point expressed as a percentage of activity, of a
specified reference score.
Particle or fibers refers to a preparation of DBM, DBM
compositions, or bone sample that has been milled, ground,
pulverized, or otherwise reduced to a particulate form. The size of
the particles or fibers is typically greater than 50 microns,
preferably greater than 75 microns, more preferably greater than
100 microns, and most preferably greater than 150 microns. These
dimensions refer to average particle diameter for more
spherical-like particles, and for particles of other shapes except
where specifically indicated it refers to the smallest
cross-sectional dimension of the particle. In certain embodiments,
the composition may include even larger sized particles, preferably
greater than 1 mm, greater than 1.5 mm, or most preferably greater
than 2 mm in their largest dimension. The particles or fibers may
be of any shape including wedges, rods, spheres, cubes, discs,
ovals, irregularly shaped, etc. For example, in certain
embodiments, the particles may be wedge-shaped and be approximately
2 mm in their largest dimension and 100 microns or less in another
dimension. The particles or fibers may be sieved or sorted in order
to collect particles of a particular size. These particles or
fibers may be mixed with a solution, slurry, deformable solid, or
liquid to form a paste to be used in administering or applying the
graft of DBM, inventive DBM composition, or bone sample. Preferred
methods of particle or fiber preparation are disclosed in issued
U.S. Pat. Nos. 5,607,269; 5,236,456; 5,284,655; 5,314,476; and
5,507,813; each of which is incorporated herein by reference.
Polysaccharide, as used herein, refers to any polymer or oligomer
of carbohydrate residues. The polymer may consist of anywhere from
two to hundreds to thousands of sugar units. Polysaccharides may be
purified from natural sources such as plants or may be synthesized
de novo in the laboratory. Polysaccharides isolated from natural
sources may be modified chemically to change their chemical or
physical properties (e.g., phosphorylated, cross-linked).
Polysaccharides may also be either straight or branch-chained. They
may contain both natural and/or unnatural carbohydrate residues.
The linkage between the residues may be the typical ether linkage
found in nature or may be a linkage only available to synthetic
chemists. Examples of polysaccharides include cellulose, maltin,
maltose, starch, modified starch, dextran, and fructose.
Glycosaminoglycans are also considered polysaccharides.
Protease inhibitors, as used herein, are chemical compounds capable
of inhibiting the enzymatic activity of protein cleaving enzymes
(i.e., proteases). The proteases inhibited by these compounds
include serine proteases, acid proteases, metalloproteases
(examples of some matrix metalloprotease inhibitors are shown in
FIG. 6), carboxypeptidase, aminopeptidase, cysteine protease, etc.
The protease inhibitor may act specifically to inhibit only a
specific protease or class of proteases, or it may act more
generally by inhibiting most if not all proteases. Preferred
protease inhibitors are protein or peptide based and are
commercially available from chemical companies such as
Aldrich-Sigma. Protein or peptide-based inhibitors which adhere to
the DBM (or calcium phosphate or ceramic carrier) are particularly
preferred as they remain associated with the matrix providing a
stabilizing effect for a longer period of time than freely
diffusible inhibitors. Examples of protease inhibitors include
aprotinin, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF),
amastatin-HCl, alpha1-antichymotrypsin, antithrombin III,
alpha1-antitrypsin, 4-aminophenylmethane sulfonyl-fluoride (APMSF),
arphamenine A, arphamenine B, E-64, bestatin, CA-074, CA-074-Me,
calpain inhibitor I, calpain inhibitor II, cathepsin inhibitor,
chymostatin, diisopropylfluorophosphate (DFP), dipeptidylpeptidase
IV inhibitor, diprotin A, E-64c, E-64d, E-64, ebelactone A,
ebelactone B, EGTA, elastatinal, foroxymithine, hirudin, leuhistin,
leupeptin, alpha2-macroglobulin, phenylmethylsulfonyl fluoride
(PMSF), pepstatin A, phebestin, 1,10-phenanthroline,
phosphoramidon, chymostatin, benzamidine HCl, antipain,
epsilon-aminocaproic acid, N-ethylmaleimide, trypsin inhibitor,
1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK),
1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), trypsin
inhibitor, and sodium EDTA.
A peptide or protein, according to the present invention, comprises
a string of at least two amino acids linked together by peptide
bonds. Inventive peptides preferably contain only natural amino
acids, although non-natural amino acids (i.e., compounds that do
not occur in nature but that can be incorporated into a polypeptide
chain) and/or amino acid analogs as are known in the art may
alternatively be employed. Also, one or more of the amino acids in
an inventive peptide may be modified, for example, by the addition
of a chemical entity such as a carbohydrate group, a phosphate
group, a farnesyl group, an isofarnesyl group, a fatty acid group,
a linker for conjugation, functionalization, or other modification,
etc.
Stabilizing agent is any chemical entity that, when included in an
inventive composition comprising DBM and/or a growth factor,
enhances the osteoinductivity of the composition as measured
against a specified reference sample. In most cases, the reference
sample will not contain the stabilizing agent, but in all other
respects will be the same as the composition with stabilizing
agent. The stabilizing agent also generally has little or no
osteoinductivity of its own and works either by increasing the
half-life of one or more of the active entities within the
inventive composition as compared with an otherwise identical
composition lacking the stabilizing agent, or by prolonging or
delaying the release of an active factor. In certain embodiments,
the stabilizing agent may act by providing a barrier between
proteases and sugar-degrading enzymes thereby protecting the
osteoinductive factors found in or on the matrix from degradation
and/or release. In other embodiments, the stabilizing agent may be
a chemical compound that inhibits the activity of proteases or
sugar-degrading enzymes. In a preferred embodiment, the stabilizing
agent retards the access of enzymes known to release and solubilize
the active factors. Half-life may be determined by immunological or
enzymatic assay of a specific factor, either as attached to the
matrix or extracted there from. Alternatively, measurement of an
increase in osteoinductivity half-life, or measurement of the
enhanced appearance of products of the osteoinductive process
(e.g., bone, cartilage or osteogenic cells, products or indicators
thereof) is a useful indicator of stabilizing effects for an
enhanced osteoinductive matrix composition. The measurement of
prolonged or delayed appearance of a strong osteoinductive response
will generally be indicative of an increase in stability of a
factor coupled with a delayed unmasking of the factor activity.
Targeting agent is any chemical entity that, when included in an
inventive compositions, will direct the composition to a particular
site or cause the inventive composition to remain in a particular
site within the recipient's body. A targeting agent may be a small
molecule, peptide, protein, biological molecule, polynucleotide,
etc. Typical targeting agents are antibodies, ligands of known
receptors, and receptors. These targeting agents may be associated
with the inventive composition through covalent or non-covalent
interactions so that the inventive composition is directed to a
particular tissue, organ, injured site, or cell type.
DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
As described herein, the present invention provides compositions
and methods relating to improved DBM or synthetic growth factor
containing compositions. Below, certain aspects of preferred
embodiments of the invention are described in more detail and with
reference to the Figures of the Drawing. Those of ordinary skill
will appreciate that a variety of embodiments or versions of the
invention are not specifically discussed below but are nonetheless
within the scope of the present invention, as defined by the
appended claims.
DBM is comprised principally of proteins and glycoproteins,
collagen being the primary protein substituent of DBM. While
collagen is relatively stable, being degraded only by the
relatively rare collagenase enzymes, the other proteins and active
factors present in DBM are quickly degraded by enzymes present in
the host. These host-derived enzymes include proteases and
sugar-degrading enzymes (e.g., endo- and exo-glycosidases,
glycanases, glycolases, amylase, pectinases, galacatosidases,
etc.). Many of the active growth factors responsible for the
osteoinductive activity of DBM exist in cryptic form, in the matrix
until activated. Activation can involve the change of a pre or pro
function of the factor, or release of the function from a second
factor or entity which binds to the first growth factor. The
instant invention alters the time course over which the active
factors present in DBM can exert their osteoinductive activity
either by 1) slowing the degradation of the active factors present
in DBM, thereby allowing them longer residence time as active
moieties, or 2) prolonging the release of one or more active
factors from the implant, or 3) altering the kinetics of activation
of one or more cryptic factors. The instant invention increases the
effective osteoinductivity of the DBM composition by (1) altering
the kinetics of activation of cryptic factors, (2) altering the
delivery and/or release of active factor from the matrix, and/or
(3) reducing proteolytic degradation of the active factor within or
as they are released from the DBM composition. Increased bone
formation presumably occurs through the recruitment of more cells
into the osteogenic phenotype.
The instant invention provides four approaches to the protection of
active factors from degradation by either host-derived or
endogenous enzymes. Factors to be protected may be endogenous to
DBM preparations or factors added to either DBM or synthetic matrix
compositions. Protection is provided through the use of a)
diffusion barriers, b) enzyme inhibitors, c) competitive
substrates, and/or d) masking moieties. These same four approaches
may be used to control the activation and/or release of
osteoinductive factors in cryptic form. For example, diffusion
barriers or activating enzyme inhibitors prevent activating enzyme
from reaching the cryptic factors or from acting upon the cryptic
factors. Preferably, degradation, release, and activation of active
factors within the DBM composition is balanced to yield a desired
osteoinductivity profile over time.
Demineralized Bone Matrix
DBM preparations have been used for many years in orthopaedic
medicine to promote the formation of bone. For example, DBM has
found use in the repair of fractures, in the fusion of vertebrae,
in joint replacement surgery, and in treating bone destruction due
to underlying disease such as rheumatoid arthritis. DBM is thought
to promote bone formation in vivo by osteoconductive and
osteoinductive processes. Osteoconduction occurs if the implanted
material serves as a scaffold for the support of new bone growth.
Osteoconduction is particularly significant when bone growth is
desired across a large or "critical size" defect, across which bone
healing would proceed only slowly or not at all. It is generally
believed that the osteoconductive properties of DBM preparations
are provided by the actual shape and coherence of the implant. Thus
DBM compositions comprising entangled fibers tend to have superior
osteoconductive properties as compared to less fibrous, more
granular preparations. Stabilizing agents which tend to preserve
the shape and/or coherence of the DBM substituent can lead to
better bone forming properties.
The osteoinductive effect of implanted DBM compositions is thought
to result from the presence of active growth factors present on the
isolated collagen-based matrix. These factors include members of
the TGF-.beta., IGF, and BMP protein families. Particular examples
of osteoinductive factors include TGF-.beta., IGF-1, IGF-2, BMP-2,
BMP-7, parathyroid hormone (PTH), and angiogenic factors. Other
osteoinductive factors such as osteocalcin and osteopontin are also
likely to be present in DBM preparations as well. There are also
likely to be other unnamed or undiscovered osteoinductive factors
present in DBM.
Any of a variety of demineralized bone matrix preparations may be
utilized in the practice of the present invention. DBM prepared by
any method may be employed including particulate or fiber-based
preparations, mixtures of fiber and particulate preparations, fully
or partially demineralized preparations, mixtures of fully and
partially demineralized preparations, including surface
demineralized preparations as described by Gertzman et al. (U.S.
Pat. No. 6,326,018, issued Dec. 4, 2001; incorporated herein by
reference). Preferred DBM compositions are described by Dowd et
al., U.S. Pat. No. 5,507,813, which is incorporated herein by
reference. Also useful are DBM preparations comprising additives or
carriers such as polyhydroxyl compounds, polysaccharides,
glycosaminoglycan proteins, nucleic acids, polymers, polaxomers,
resins, clays, calcium salts, and/or derivatives thereof.
In certain embodiments, the DBM material utilized to formulate
inventive compositions has greater than 50%, preferably greater
than 75%, more preferably greater than 80%, 85%, 90%, or 95% and
most preferably greater than 98% of the calcium phosphate removed.
The bone used in creating the DBM may be obtained from any source
of living or dead tissue. Often, it will be preferred that the
source of bone be matched to the eventual recipient of the
inventive composition. At a minimum, it is often desirable that the
donor and recipient are of the same species, though even xenogenic
sources are permitted.
Once a bone sample is obtained, it is milled, ground, pulverized,
or otherwise reduced to particulate form. In preferred embodiments,
the particles will be greater than 75 microns in their minimum
dimension, more preferably greater than 100 microns, and more
preferably greater than 150 microns. However, it should be noted
that one method of the preferred invention is to stabilize implants
containing particles less than 100 microns in any dimension and
potentially even less than 75 microns. Particles of 75 microns or
less, following demineralization, are known to have limited or no
osteoinductivity, and aspects of the present invention may be used
to enhance the activity of these small size particles as well. For
preparations employing DBM of these small sizes, at least one
stabilizing agent is used which retards the influx of host cells
capable of removing such small particles (e.g., macrophages and
foreign body giant cells) long enough to allow the active factors
within the DBM to elicit an osteoinductive response. In addition or
alternatively, a diffusion barrier will be present to retard the
efflux of factors from the particles. In certain embodiments, the
particles are at least 200 microns across the greatest dimension.
The particles may be any shape including ovals, spherical,
cuboidal, cones, pyramids, wedges, etc. In certain embodiments, the
particles are wedges, pyramids, or cones being 200 microns across
their largest dimension. In other embodiments, the DBM composition
may include a mixture of several different sizes and/or shapes of
particles.
Following particulation, the DBM is treated to remove mineral from
the bone. While hydrochloric acid is the industry-recognized
demineralization agent of choice, the literature contains numerous
reports of methods for preparing DBM (see, for example, Russell et
al. Orthopaedics 22(5):524-531, May 1999; incorporated herein by
reference). For the purposes of the present invention, any material
that provides a scaffolding containing active osteoinductive
factors is considered DBM. The DBM may be prepared by methods known
in the art or by other methods that can be developed by those of
ordinary skill in the art without undue experimentation. In some
instances, large fragments or even whole bone may be demineralized,
and then particulated following demineralization. DBM prepared in
this way is within the scope of the invention.
In the preparing the improved DBM compositions, the DBM component
may be ground or otherwise processed into particles of an
appropriate size before or after demineralization. In certain
embodiments, the particle size is greater than 75 microns, more
preferably ranging from about 100 to about 3000 microns, and most
preferably from about 200 to about 2000 microns. After grinding the
DBM component to the desired size, the mixture may be sieved to
select those particles of a desired size. In certain embodiments,
the DBM particles may be sieved though a 50 micron sieve, more
preferably a 75 micron sieve, and most preferably a 100 micron
sieve.
One particularly useful way to protect small size particles from
cellular ingestion and/or provide a diffusion barrier is to embed
them in a monolithic bioabsorbable matrix, and then fragment the
particle-containing monolithic matrix into particle sizes greater
than 70 microns, preferably greater than 100 microns, and most
preferably greater than 150 microns in their smallest dimension.
Preferred matrices for embedding small DBM particles include
biocompatible polymers and setting calcium phosphate cements.
Generally the particulate DBM/polymer weight ratio will range from
about 1:5 to about 1:3. In the case of calcium phosphate, the DBM
will be present up to 75% by weight. Particulation of the monolith
can be accomplished by conventional milling or grinding, or through
the use of cryomilling, or freezing followed by pulverization. In
one preferred embodiment, lyophilized DBM is embedded in a
resorbable polymer. In a second preferred embodiment, lyophilized
DBM is embedded in one of the setting calcium phosphates known to
the art.
Stabilizing Agents
Diffusion barriers. Diffusion barriers retard the diffusion of
degradative enzymes and/or water to the active moieties within the
inventive formulations. Enzymes retarded in their diffusion to the
included DBM may be capable of releasing the active factor from the
matrix, and/or degrading or inactivating the active factor. They
also may act by retarding diffusion of the active factors from the
implant site. In these ways, the barriers provide for longer
residence time of the active factors at the implant site. This is
particularly useful for forming bone in higher species such as
humans, where bone formation appears to require the presence of
active factors for longer times.
Generally, materials most suitable to serve as diffusion barriers
will be easily mixed with DBM or synthetic matrix of choice to form
a gel, paste, or putty-like consistency, although in some
embodiments, the barrier/matrix formulation will be prepared as a
relatively non-deformable solid (e.g., for matrix preparations to
be used in posterior lateral spine fusion). In preferred
embodiments, the diffusion barriers themselves degrade in a
predictable manner to unmask active factors at a time later than
would normally occur in the absence of a diffusion barrier.
Resorbable polymers with known hydrolytic rates are useful as
diffusion barriers as well as enzymatically degraded polymers.
Particularly useful are lipase susceptible lipid based carriers
such as fatty acids and phospholipids, which mix well with DBM. In
certain DBM embodiments, the composition does not include
phosphatidylcholine. Some particularly effective preparations
provide prolonged stability by controlled unmasking of the
osteoinductive factors. These preparations generally involve the
use of two or more diffusion barriers with different degradation
times affording at least two different rates of unmasking the same
active factor.
Biodegradable polymers useful in preparing inventive stabilized
matrix/growth factor compositions include natural polymers such as
proteins (e.g., collagen) and polysaccharides (e.g., starch,
modified starch, maltrin) as well as man-made resorbable polymers
such as poly-orthoesters. These polymers when mixed with the
inventive growth factor containing compositions retard diffusion of
the host's degradative enzymes and/or water to the active factors
contained within the composition, thereby retarding release and/or
degrading of the active factor contained therein.
Polymers that may be included within inventive compositions
include, for example, natural polymers such as lipids,
polysaccharides, proteoglycans, and proteins. Preferred
polysaccharides include starches, dextrans, and celluloses, and
preferred proteins include collagen. Polysaccharides such as
starches, dextrans, and celluloses may be unmodified or may be
modified physically or chemically to affect one or more of their
properties such as their characteristics in the hydrated state,
their solubility, their susceptibility to degradation, or their
half-life in vivo. Polysaccharides such as starches and celluloses
are attractive as they also have known degradation rates.
Generally, the celluloses degrade more slowly within the body,
breaking down on the order of weeks or months, while many starch
and lipid preparations degrade rapidly, on the order of hours or
days. Starch in the natural state is a mixture of two
polysaccharides, amylose and amylopectin. The susceptibility of the
particular starch to the starch-degrading enzymes such as amylase,
pectinases, and .beta.-glucosidase is an important consideration in
designing the inventive formulations. Those skilled in the art are
aware of the variety of amylase susceptibilities of starches
prepared from various plant sources and may apply this knowledge to
produce formulations having a desired stability time. Preferred
starches will degrade as much as 10% per day, preferably 50% per
day, and most preferably greater than 90% per day. Those starches
less susceptible to degradation by pectinase and/or amylase
(amylase-resistant starch; Starch Australasia, Sydney, Australia)
may be used to maximally extend the osteoinductive half-life in
vivo to an even greater extent than improved DBM or synthetic
growth factor/matrix formulations prepared from more enzyme
susceptible starches. Some modified starches are less susceptible
to degradation by amylase; therefore, improved DBM with modified
starch would presumably have a longer half-life in vivo as compared
to those improved DBM with unmodified starch. One preferred method
to affect amylase susceptibility of starch is through the use of
starch lipid combinations. Guidance for the combination of lipid
and starch to affect amylase susceptibility is given by Crowe et al
"Inhibition of Enzymic Digestion of Amylose by Free Fatty Acids In
Vitro Contributes to Resistant Starch Formation" J. Nutr.
130(8):2006-2008, August 2000; incorporated herein by reference.
Similar considerations apply to lipids and their degradative
enzymes the lipases. A large variety of mono-, di-, and
triglycerides with varying degrees of susceptibility to lipase
degradation are available from commercial sources. Some embodiments
include one or more polymeric materials, preferably biodegradable,
such as tyrosine polycarbonates, polyfumarates, tyrosine
polyarylates, and poly-orthoesters such as polylactide,
polygalactide, and co-polymers thereof. These polymers are
biodegradable, and their properties can be modified by altering the
chain length or degree of cross-linking of the polymer and/or the
chemical structure of the monomers. Additionally, co-polymers can
be prepared using combinations of resorbable polymers.
Enzyme inhibitors. Alternatively or additionally, the inventive
compositions may be stabilized by the addition of one or more
degradation inhibitors, active against growth factor activity
degrading agents found in the host organism and/or in the implant
composition. These inhibitors may also inhibit the activity of
enzymes responsible activating osteoinductive factors of the DBM
composition. Degradation or activation inhibitors useful in the
practice of the present invention may include, for example, acid
protease inhibitors, serine protease inhibitors, metalloprotease
inhibitors (shown in FIG. 6; also, see Whittaker et al. "Matrix
Metalloproteinases and their Inhibitors-Current Status and Future
Challenges" Celltransmissions 17(1):3-14; incorporated herein by
reference), cysteine protease inhibitors, glyconase inhibitors, and
glycosidase inhibitors. Specific protease inhibitors useful in the
practice of the present invention include, for example, aprotinin,
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), amastatin-HCl,
alpha1-antichymotrypsin, antithrombin III, alpha1-antitrypsin,
4-aminophenylmethane sulfonyl-fluoride (APMSF), arphamenine A,
arphamenine B, E-64, bestatin, CA-074, CA-074-Me, calpain inhibitor
I, calpain inhibitor II, cathepsin inhibitor, chymostatin,
diisopropylfluorophosphate (DFP), dipeptidylpeptidase IV inhibitor,
diprotin A, E-64c, E-64d, E-64, ebelactone A, ebelactone B, EGTA,
elastatinal, foroxymithine, hirudin, leuhistin, leupeptin,
alpha2-macroglobulin, phenylmethylsulfonyl fluoride (PMSF),
pepstatin A, phebestin, 1,10-phenanthroline, phosphoramidon,
chymostatin, benzamidine HCl, antipain, epsilon-aminocaproic acid,
N-ethylmaleimide, trypsin inhibitor,
1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK),
1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), trypsin
inhibitor, sodium EDTA, and the TIMPs class of metalloproteinase
inhibitors. Particularly useful ones are those stable under acidic
conditions and effective at acidic conditions. As will be
appreciated by those of skill in this art, the less osteoinductive
factors lost or degraded during the processing of the bone to form
DBM the more will be available for recruitment once the DBM
composition is implanted.
In some embodiments, a composition for implantation is provided
comprising demineralized bone matrix and an excipient or
stabilizing agent. Such composition for implantation may exhibit an
osteoinductivity that is at least about 10% greater than
osteoinductivity of a composition of demineralized bone matrix
without excipient. The excipient may be, for example, a diffusion
barrier, an enzyme inhibitor, a competitive substrate, a masking
entity, a polymer (natural, non-natural, modified, or derivated),
growth factor binding protein, lectin, antibody, other material,
and combinations thereof. Various suitable excipients are discussed
more fully below. These excipients are intended to be illustrative
only. Further, in some embodiments, the demineralized bone matrix
may be embedded in the excipient.
Competitive substrates. Use of competitive substrates for the
host's degradative or activating enzymes may also be employed to
stabilize the osteoinductive factors of the DBM or exogenously
added growth factors. Examples of competitive substrates include
di- and poly-lysines. Di- and polysaccharides can be employed as
competitive substrates of glycosidases, amylases, and/or
pectinases. Particularly useful are stereoisomers of the
competitive substrates.
Masking entities. Specific masking entities are generally used to
specifically block a single entity or class of entities from
enzymatic breakdown. The degradative or activating enzyme to be
blocked may be endogenous or exogenous to the matrix. The masking
entities generally bind to a ligand present on the matrix which may
or may not be the active factor itself. Once bound the masking
entity sterically hinders the breakdown and/or release of one or
more active factors. Over time the masking entity either unbinds or
itself is degraded leaving the ligand and or growth factor
susceptible to degradation. Diffusion barriers represent a
generalized form of masking entity by preventing access of the
degradative or activating enzymes to many or all the growth factors
associated with the matrix.
Growth factor binding proteins: Virtually every extracellular
matrix growth factor is know to be associated with a binding
protein which regulates the activity of the growth factor. Purified
preparations of these binding proteins can be prepared, and added
to DBM preparations to serve as masking entities. Typical growth
factor binding proteins include but are not limited to noggin,
chordin, follistatin, TGF-.beta. binding protein, and insulin-like
growth factor binding proteins. Agents may also be added to the DBM
composition to induce the release of the growth factor from its
binding protein. In certain embodiments, the agent known to induce
release of the growth factor may be encapsulated in a biodegradable
polymer so that the agent is released over an extended period of
time, thereby leading to the release of growth factor over an
extended period of time.
Lectins. Lectins are proteins which can bind to the sugar moieties
of glycoproteins. Since growth factors are generally glycoproteins,
lectins can be employed to bind to the growth factors and
potentially retard or inhibit access of proteases or growth factor
releasing enzymes to the active growth factors. Ideally the lectin
will be selected according to the identity of the terminal sugar on
the active glycoprotein of interest. Lectins include, but are not
limited to, membrane-bound lectins, I-type lectins, and P-type
lectins. Specific lectins include galectins, calcium-dependent
lectins, selecting, collecting, and annexins.
Antibodies. Monoclonal or polyclonal antibodies specific to the
active factors, or to those proteins known to bind to the active
factors (see above) may be added to the inventive formulations to
protect specific growth factors from degradative or releasing
enzymes.
Inventive DBM compositions may alternatively or additionally be
stabilized through exposure to conditions (e.g., pH, temperature,
etc.) under which degrading agents do not function optimally or the
degradatory enzymes will not function effectively (e.g., low
pH).
Addition of enzyme inhibitors, competitive substrates, and masking
agents. The incorporation of any of these entities into the
inventive formulations, is generally accomplished by suspending the
molecule or molecules of interest in an appropriately compatible
buffer as will be known to those skilled in the art. This buffer
will be mixed with lyophilized matrix in a relatively low
liquid-to-solid volume ratio to form a slurry. The slurry is then
lyophilized and used to prepare the desired DBM formulations.
One unexpected feature of the instant invention is that the
incorporation of any of the inventive enzyme inhibitors,
competitive substrates, or masking agents often has the additional
feature of improving the DBM formulation shelf-life by preventing
access of endogenously present degradative enzymes to the active
factors present in the matrix. This is particularly true for DBM
formulations which are prepared containing water (e.g., DBM
preparations with hydrogel carriers such as hyaluronic acid or
collagen, or hydrated starch carriers).
Many of the osteoinductive factors found in DBM are in cryptic form
and must be "activated" or "released" in order to be
osteoinductive. The activation of osteoinductive factors may
involve a conformational change, a post-translational modification,
a cleavage of the peptide, a change in tertiary or quaternary
structure, release from the DBM, release from a binding protein,
etc. For example, the factors may be in a pre- or pro-form which
requires proteolytic cleavage to be active. In addition, the
osteoinductive factors may be associated with a binding protein or
a protein of the matrix of the DBM. The same processes such as
proteolysis involved in degradation of the active factors may also
be involved in the activation of these factors. Therefore, all the
same methods described above that can be used to slow degradation
may also affect activation rates. One of skill in the art preparing
a DBM composition could balance the rates of degradation and
activation to achieve a desired level of osteoinductivity from the
implant over time. In addition, such factors as pH, ion
concentration, or other factors which affect protein function
and/or folding may affect the activation of osteoinductive factors
found in DBM. These factors also may effect the release of a factor
from its binding protein. In certain embodiments, for example,
where pH plays a role in the activation of a factor, the DBM
composition may include a chemical compound such as a polymer which
will break down over time and release an acid by-product; thereby,
activating the factors within the DBM composition. In other
embodiments, a biodegradable polymer may release ions or a protease
that is able to "activate" the osteoinductive factors of the DBM
composition.
Release of the osteoinductive factors from the delivery matrix may
also be important in its osteoinductivity. Many factors may be
found bound to the DBM through specific binding proteins as
described above or through non-specific interactions. A portion of
the factors may need to be released from the matrix in order to be
active while others may only be active while bound. For example,
cells may be recruited to the matrix by certain factors, and then
once there, the cells may interact with other factors bound to the
matrix. The cells may need to interact with both the matrix and the
factor to induce bone production. The rate of release of the
osteoinductive factors may be controlled by diffusion barriers or
agents which affect the binding of the factors to the matrix or
their binding proteins. As described above, in certain embodiments,
it is preferred that a diffusion barrier be degraded over time so
as to release factors or allow recruited cells to interact with the
matrix. Degradation of the diffusion barrier may also allow
proteases into the DBM implant to activate and/or release
osteoinductive factors.
As will be appreciated by one of skill in this art, the DBM
composition may be prepared to balance degradation, activation, and
release of osteoinductive factors to create a composition with a
desired osteoinductive activity. The osteoinductivity of the DBM
composition may be suited for a particular application, site of
implant, or patient. For instance, certain application would
require an extended period of osteoinductivity ranging from weeks
to months; whereas other applications may only need
osteoinductivity for days to weeks. One of skill in the art can
prepare a DBM composition with a desired osteoinductivity time
profile.
Test for Enhancement
The invention also provides a simple in vitro test for the
screening of suitable stabilizing agents. DBM prepared with and
without the biodegradable stabilizing agent is exposed under
simulated physiological conditions (e.g., pH 7.4, physiological
saline) to an enzyme or combination of enzymes known to be capable
of degrading some or all of the protein constituents of the DBM.
Most often this will be a protease such as trypsin, papain,
peptidase, or the like. Evidence for matrix or matrix component
breakdown is compared between the two preparations. Materials
retarding the breakdown process are considered to be good
candidates for further testing. Preferred indicators of breakdown
include immunological detection of TGF-.beta. and/or IGF breakdown.
In addition to the enzymes indicated above, other enzymes such as
collagenases or combinations of enzymes as well as glycosidases may
also be used. Particularly useful in this regard is the natural
degradatory activity of serum or tissue extracts. Under these
conditions, specific marker proteins present in the DBM may be
tracked by immunological methods such as radioimmunoassay or gel
electrophoresis utilizing western blots, or other analytical
methods known in the art.
Following the identification of candidate stabilizers in the above
assay, the DBM formulations containing the candidate stabilizers
are tested in the osteoinductivity assays described elsewhere
herein.
Osteoinducer
To the improved DBM may be added other osteoinducing agents. These
agents may be added in an activated or non-activated form. These
agents may be added at anytime during the preparation of the
inventive material. For example, the osteoinducing agent may be
added after the demineralization step and prior to the addition of
the stabilizing agents so that the added osteoinducing agent is
protected from exogenous degrading enzymes once implanted. In some
embodiments the DBM is lyophilized in a solution containing the
osteoinducing agent. In certain other preferred embodiments, the
osteoinducing agents are adhered onto the hydrated demineralized
bone matrix and are not freely soluble. In other instances, the
osteoinducing agent is added to the improved DBM after addition of
the stabilizing agent so that the osteoinducing agent is available
immediately upon implantation of the DBM.
Osteoinducing agents include any agent that leads to or enhances
the formation of bone. The osteoinducing agent may do this in any
manner, for example, the agent may lead to the recruitment of cells
responsible for bone formation, the agent may lead to the secretion
of matrix which may subsequently undergo mineralization, the agent
may lead to the decreased resorption of bone, etc. Particularly
preferred osteoinducing agents include bone morphogenic proteins
(BMPs), transforming growth factor (TGF-.beta.), insulin-like
growth factor (IGF-1), parathyroid hormone (PTH), and angiogenic
factors such as VEGF. In one preferred embodiment (Example 12), the
inducing agent is genetically engineered to comprise an amino acid
sequence which promotes the binding of the inducing agent to the
DBM or the carrier. Sebald et al. in PCT/EP00/00637, incorporated
herein by reference, describe the production of exemplary
engineered growth factors, suitable for use with DBM.
Formulation
Improved osteogenic compositions of the present invention may be
formulated for a particular use. The formulation may be used to
alter the physical, biological, or chemical properties of a DBM
preparation. A physician would readily be able to determine the
formulation needed for a particular application taking into account
such factors as the type of injury, the site of injury, the
patient's health, the risk of infection, etc.
Inventive compositions therefore may be prepared to have selected
resorption/loss of osteoinductivity rates, or even to have
different rates in different portions of an implant. For example,
the formulation process may include the selection of DBM particles
of a particular size or composition, combined with the selection of
a particular stabilizing agent or agents, and the amounts of such
agents. To give but one example, it may be desirable to provide a
composition whose osteoinductive factors are active in a relatively
constant amount over a given period of time. A DBM composition
comprising factors with longer half-lives can be prepared using a
less biodegradable polymer or a larger amount (e.g., a thicker
coating) of polymeric compound. Alternatively or additionally, the
particle size may be important in determining the half-life of the
inventive DBM composition. In certain preferred embodiments, an
inventive formulation may include a mixture of particles, each with
a different half-life. Such a mixture could provide the steady or
possible unmasking of osteoinductive factors over an extended
period of time ranging from days to weeks to months depending on
the needs of the injury. Compositions such as this can be
formulated to stimulate bone growth in a human patient comparable
to the bone growth induced by treatment with 10 .mu.g of rhBMP on a
collagen sponge, and preferably comparable to 100 .mu.g, and most
preferably 1-10 mg rhBMP.
Physical properties such as deformability and viscosity of the DBM
may also be chosen depending on the particular clinical
application. The particles of the improved DBM may be mixed with
other materials and factors to improve other characteristics of the
implant. For example, the improved DBM material may be mixed with
other agents to improve wound healing. These agents may include
drugs, proteins, peptides, polynucleotides, solvents, chemical
compounds, biological molecules.
The particles of DBM (or inventive DBM material) may also be formed
into various shapes and configurations. The particles can be formed
into rods, strings, sheets, weaves, solids, cones, discs, fibers,
wedges etc. In certain embodiments, the shape and size of the
particles in the DBM composition affect the time course of
osteoinductivity. For example, in a cone or wedge shape, the
tapered end will result in osteoinductivity shortly after
implantation of the DBM composition, whereas the thicker end will
lead to osteoinductivity later in the healing process (e.g., hours
to days to weeks later). In certain embodiments, the particle have
a length of greater than 2 mm, greater than 1.5 mm, greater than 1
mm, preferably greater than 500 microns, and most preferably
greater than 200 microns across its widest dimension. Also, larger
particle size will have induce bone formation over a longer time
course than smaller particles. Particles of different
characteristics (e.g., composition, size, shape) may be used in the
formation of these different shapes and configurations. For
example, in a sheet of DBM a layer of long half-life particles may
be alternated between layers of shorter half-life particles (See
U.S. Pat. No. 5,899,939, incorporated herein by reference). In a
weave, strands composed of short half-life particles may be woven
together with strands of longer half-lives.
In one preferred embodiment of the invention, fibrous DBM is shaped
into a matrix form as described in U.S. Pat. No. 5,507,813,
incorporated herein by reference, and Examples 13 & 14
(embedded matrix fabrication) below. The shaped DBM is then
embedded within a diffusion barrier type matrix, such that a
portion of the matrix is left exposed free of the matrix material.
Particularly preferred blocking matrices are starch, phosphatidyl
choline, tyrosine polycarbonates, tyrosine polyarylates,
polylactides, polygalactides, or other resorbable polymers or
copolymers. Devices prepared in this way from these matrices have a
combination of immediate and longer lasting osteoinductive
properties and are particularly useful in promoting bone mass
formation in human posterolateral spine fusion indications.
In another embodiment of the invention, inventive DBM compositions
having a pre-selected three-dimensional shape are prepared by
repeated application of individual layers of DBM, for example by
3-D printing as described by Cima et al. U.S. Pat. Nos. 5,490,962;
and 5,518,680, each of which is incorporated herein by reference;
and Sachs et al. U.S. Pat. No. 5,807,437, incorporated herein by
reference. Different layers may comprise individual stabilized DBM
preparations, or alternatively may comprise DBM layers treated with
stabilizing agents after deposition of multiple layers.
In the process of preparing improved inventive DBM materials, the
materials may be produced entirely aseptically or be sterilized to
eliminate any infectious agents such as HIV, hepatitis B, or
hepatitis C. The sterilization may be accomplished using
antibiotics, irradiation, chemical sterilization (e.g., ethylene
oxide), or thermal sterilization. Other methods known in the art of
preparing DBM such as defatting, sonication, and lyophilization may
also be used in preparing the improved DBM. Since the biological
activity of demineralized bone is known to be detrimentally
affected by most terminal sterilization processes, care must be
taken when sterilizing the inventive compositions. In preferred
embodiments, the DBM compositions described herein will be prepared
aseptically or sterilized as described in Example 11.
Applications
Improved osteogenic compositions of the present invention may be
used to promote the healing of bone injuries. The compositions may
be used in any bone of the body on any type of injury. The improved
DBM composition has been designed to produce bone in human patients
with similar timing and at a level similar to 10 .mu.g to 100
.mu.g, preferably 200 .mu.g to 1 mg of rhBMP on a collagen sponge.
For example, specific bones that can be repaired using the
inventive material include the ethmoid, frontal, nasal, occipital,
parietal, temporal, mandible, maxilla, zygomatic, incus, stapes,
malleus, cervical vertebrae, thoracic vertebrae, lumbar vertebrae,
sacrum, sternum, ribs, clavicle, scapula, humerus, ulna, radius,
carpal bones, metacarpal bones, phalanges, ileum, ischium, pubis,
pelvis, femur, patella, tibia, fibula, calcaneus, talus, and
metatarsal bones. The type of injury amenable to treatment with the
improved DBM include bone defects resulting from injury, brought
about during the course of surgery, infection, malignancy, or
developmental malformation. The inventive material may be useful in
orthopaedic, neurosurgical, cosmetic, and oral and maxillofacial
surgical procedures such as the repair of simple and compound
fractures and non-unions, external and internal fixations, joint
reconstructions such as arthrodesis, general arthroplasty, cup
arthroplasty of the hip, femoral and humeral head replacement,
femoral head surface replacement and total joint replacement,
repairs of the vertebral column including spinal fusion and
internal fixation, tumor surgery (e.g., deficit filling),
discectomy, laminectomy, excision of spinal cord tumors, anterior
cervical and thoracic operations, repair of spinal injuries,
scoliosis, lordosis and kyphosis treatments, intermaxillary
fixation of fractures, mentoplasty, temporomandibular joint
replacement, alveolar ridge augmentation and reconstruction, inlay
bone grafts, implant placement and revision, sinus lifts, etc.
Inventive DBM compositions may also be used as drug delivery
devices. In certain preferred embodiments, association with the
inventive DBM composition increases the half-life of the relevant
biologically active agent(s). Particularly preferred inventive drug
delivery devices are used to deliver osteoinductive growth factors.
Other preferred agents to be delivered include factors or agents
that promote wound healing. However, inventive compositions may
alternatively or additionally be used to deliver other
pharmaceutical agents including antibiotics, anti-neoplastic
agents, growth factors, hematopoietic factors, nutrients, etc.
Bioactive agents that can be delivered using the inventive DBM
composition include non-collagenous proteins such as osteopontin,
osteonectin, bone sialo proteins, fibronectin, laminin, fibrinogen,
vitronectin, trombospondin, proteoglycans, decorin, proteoglycans,
beta-glycan, biglycan, aggrecan, veriscan, tenascin, matrix gla
protein hyaluronan; cells; amino acids; peptides; inorganic
elements; inorganic compounds; organometallic compounds; cofactors
for protein synthesis; cofactors for enzymes; vitamins; hormones;
soluble and insoluble components of the immune system; soluble and
insoluble receptors including truncated forms; soluble, insoluble,
and cell surface bound ligands including truncated forms;
chemokines, interleukins; antigens; bioactive compounds that are
endocytosed; tissue or tissue fragments; endocrine tissue; enzymes
such as collagenase, peptidases, oxidases, etc.; polymeric cell
scaffolds with parenchymal cells; angiogenic drugs, polymeric
carriers containing bioactive agents; encapsulated bioactive
agents; bioactive agents in time-release form; collagen lattices;
antigenic agents; cytoskeletal agents; cartilage fragments; living
cells such as chondrocytes, osteoblasts, osteoclasts, fibroblasts,
bone marrow cells, mesenchymal stem cells, etc.; tissue
transplants; bioadhesives; bone morphogenic proteins (BMPs),
transforming growth factor (TGF-beta), insulin-like growth factor
(IGF-1, IGF-2), platelet derived growth factor (PDGF); fibroblast
growth factors (FGF), vascular endothelial growth factor (VEGF),
epidermal growth factor (EGF), growth factor binding proteins,
e.g., insulin-like growth factor binding protein (IGFBP-2, IGFBP-3,
IGFBP-4, IGFBP-5, IGFBP-6); angiogenic agents; bone promoters;
cytokines; interleukins; genetic material; genes encoding bone
promoting action; cells containing genes encoding bone promoting
action; cells genetically altered by the hand of man; externally
expanded autograft or xenograft cells; growth hormones such as
somatotropin; bone digestors; antitumor agents; fibronectin;
cellular attractants and attachment agents; immunosuppressants;
bone resorption inhibitors and stimulators; mitogenic factors;
bioactive factors that inhibit and stimulate second messenger
molecules; cell adhesion molecules, e.g., cell-matrix and cell-cell
adhesion molecules; secondary messengers; monoclonal antibodies
specific to cell surface determinants on mesenchymal stem cells;
portions of monoclonal antibodies specific to cell surface
determinants on mesenchymal stem cells; clotting factors;
polynucleotides; and combinations thereof. The amount of the
bioactive agent included with the DBM composition can vary widely
and will depend on such factors as the agent being delivered, the
site of administration, the patient's physiological condition, etc.
The optimum levels being determined in a specific case based upon
the intended use of the implant.
For example, inventive DBM compositions may be prepared so that
they include one or more compounds selected from the group
consisting of drugs that act at synaptic and neuroeffector
junctional sites (e.g., acetylcholine, methacholine, pilocarpine,
atropine, scopolamine, physostigmine, succinylcholine, epinephrine,
norepinephrine, dopamine, dobutamine, isoproterenol, albuterol,
propranolol, serotonin); drugs that act on the central nervous
system (e.g., clonazepam, diazepam, lorazepam, benzocaine,
bupivacaine, lidocaine, tetracaine, ropivacaine, amitriptyline,
fluoxetine, paroxetine, valproic acid, carbamazepine,
bromocriptine, morphine, fentanyl, naltrexone, naloxone,); drugs
that modulate inflammatory responses (e.g., aspirin, indomethacin,
ibuprofen, naproxen, steroids, cromolyn sodium, theophylline);
drugs that affect renal and/or cardiovascular function (e.g.,
furosemide, thiazide, amiloride, spironolactone, captopril,
enalapril, lisinopril, diltiazem, nifedipine, verapamil, digoxin,
isordil, dobutamine, lidocaine, quinidine, adenosine, digitalis,
mevastatin, lovastatin, simvastatin, mevalonate); drugs that affect
gastrointestinal function (e.g., omeprazole, sucralfate);
antibiotics (e.g., tetracycline, clindamycin, amphotericin B,
quinine, methicillin, vancomycin, penicillin G, amoxicillin,
gentamicin, erythromycin, ciprofloxacin, doxycycline, acyclovir,
zidovudine (AZT), ddC, ddI, ribavirin, cefaclor, cephalexin,
streptomycin, gentamicin, tobramycin, chloramphenicol, isoniazid,
fluconazole, amantadine, interferon,); anti-cancer agents (e.g.,
cyclophosphamide, methotrexate, fluorouracil, cytarabine,
mercaptopurine, vinblastine, vincristine, doxorubicin, bleomycin,
mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin,
decarbazine); immunomodulatory agents (e.g., interleukins,
interferons, GM-CSF, TNF.alpha., TNF.beta., cyclosporine, FK506,
azathioprine, steroids); drugs acting on the blood and/or the
blood-forming organs (e.g., interleukins, G-CSF, GM-CSF,
erythropoietin, vitamins, iron, copper, vitamin B.sub.12, folic
acid, heparin, warfarin, coumarin); hormones (e.g., growth hormone
(GH), prolactin, luteinizing hormone, TSH, ACTH, insulin, FSH, CG,
somatostatin, estrogens, androgens, progesterone,
gonadotropin-releasing hormone (GnRH), thyroxine,
triiodothyronine); hormone antagonists; agents affecting
calcification and bone turnover (e.g., calcium, phosphate,
parathyroid hormone (PTH), vitamin D, bisphosphonates, calcitonin,
fluoride), vitamins (e.g., riboflavin, nicotinic acid, pyridoxine,
pantothenic acid, biotin, choline, inositol, camitine, vitamin C,
vitamin A, vitamin E, vitamin K), gene therapy agents (e.g., viral
vectors, nucleic-acid-bearing liposomes, DNA-protein conjugates,
anti-sense agents); or other agents such as targeting agents
etc.
In certain embodiments, the agent to be delivered is adsorbed to or
otherwise associated with the matrix being implanted. The agent may
be associated with the matrix of the DBM composition through
specific or non-specific interactions; or covalent or non-covalent
interactions. Examples of specific interactions include those
between a ligand and a receptor, a epitope and an antibody, etc.
Examples of non-specific interactions include hydrophobic
interactions, electrostatic interactions, magnetic interactions,
dipole interactions, van der Waals interactions, hydrogen bonding,
etc. In certain embodiments, the agent is attached to the matrix
using a linker so that the agent is free to associate with its
receptor or site of action in vivo. In certain preferred
embodiments, the agent to be delivered may be attached to a
chemical compound such as a peptide that is recognized by the
matrix of the DBM composition. In another embodiment, the agent to
be delivered is attached to an antibody, or fragment thereof, that
recognizes an epitope found within the matrix of the DBM
composition. In a particularly preferred embodiment, the agent is a
BMP, TGF-.beta., IGF, parathyroid hormone (PTH), growth factors, or
angiogenic factors. In certain embodiments at least two bioactive
agents are attached to the DBM composition. In other embodiments at
least three bioactive agents are attached to the DBM
composition.
The growth factor stabilizing strategies described herein, may also
be applied directly to growth factors associated with synthetic
matrices such as ceramics, bone cements, or polymers. In these
embodiments one, two, or more growth factors are associated with
the synthetic matrix. A growth factor is associated with an
anchoring matrix (e.g., amorphous or crystalline calcium phosphate
associated with a growth factor such as BMP), wherein the
composition is prepared in the presence of a diffusion barrier such
as amylose, fatty acid, or a resorbable polymer, or with a
combination of at least two or more of these stabilizing agents. In
a preferred embodiment, a poorly crystalline calcium phosphate is
associated with a growth factor mixed with a starch/lecithin
diffusion barrier.
These and other aspects of the present invention will be further
appreciated upon consideration of the following Examples, which are
intended to illustrate certain particular embodiments of the
invention but are not intended to limit its scope, as defined by
the claims.
EXAMPLES
Example 1
Preparing Demineralized Bone Matrix (DBM)
DBM may be prepared using any method or technique known in the art
(see Russell et al. Orthopedics 22(5):524-531, May 1999;
incorporated herein by reference). The following is an exemplary
procedure for preparing demineralized bone derived from Glowacki et
al. "Demineralized Bone Implants" Clinics in Plastic Surgery
12(2):233-241, April 1985, which is incorporated herein by
reference. Bones or bone fragments from donors are cleaned to
remove any adherent periosteum, muscle, connective tissue, tendons,
ligaments, and cartilage. Cancellous bone may be separated from
dense cortical bone and processed as large pieces. Cortical bone
may be cut into small pieces to improve the efficiency of
subsequent washes and extractions. Denser bone from larger animals
may need to be frozen and hammered in order to produce chips less
than 1 cm. The resulting pieces of bone are thoroughly washed with
cold, deionized water to remove marrow and soft tissue.
The cleaned bone is then extracted with frequent changes of
absolute ethanol for at least 1 hour. Typically, a total of 4
liters of ethanol is used per 100 g of bone. The bone is then
extracted with frequent changes of anhydrous diethyl ether in a
fume hood for 1 hour. Typically, 2 liters of ether is used per 100
g of bone. The bone is dehydrated by these extractions of ethanol
and ether and can be stored at room temperature.
The dehydrated bone is then frozen and then pulverized in a liquid
nitrogen impacting mill. Pulverized bone is then sieved into
fractions of 75 to 250, 250 to 450, and greater than 450 microns.
Bone particle fractions are then demineralized using 0.5 M
hydrochloric acid (50 ml per gram) for 3 hours at room temperature
or at 4 .degree. C. on magnetic stirrers with insulation to prevent
overheating. Large chips of bone and blocks are extracted
completely at 4 .degree. C. with frequent changes of 0.5 M
hydrochloric acid. The demineralization process can be monitored
radiographically, by ashing, or by nondecalcified histologic
techniques (von Kossa stain). The acid and liberated minerals are
washed away with cold, deionized water until the pH of the wash
matches the pH of the water. The water washes can be decanted from
the large particles and chips of bone; however, the washes must be
removed by centrifugation from the finer particles. The washing
step requires approximately 500 ml of water per gram of starting
bone particles.
Demineralized bone powders are extracted with changes of absolute
ethanol for 1 hour using 200 ml of ethanol per gram of starting
bone particles. The material is extracted in a fume hood with
changes of anhydrous ethyl ether for 1 hour with 100 ml of ether
per gram of starting bone particles. After the last change of ether
is removed, the demineralized bone powder is left overnight in the
hood until all the residual ether has vaporized. The particles
should be odorless, snow-white, and discrete. To sterilize the
demineralized bone material, it may be treated with cold ethylene
oxide or irradiated.
To test the bioactivity of the prepared DBM, 25 mg of the material
is implanted into each of two thoracic subcutaneous pockets in
shaved, anesthetized 28-day old male Charles River CD rats. The
implanted specimens may then be harvested and inspected several
days after implantation. The composition of the induced tissue can
be quantified by histomorphometric analysis and be biochemical
techniques.
Example 2
Another Method of Preparing DBM
DBM may be prepared using any method or techniques known in the art
(See Russell et al. Orthopedics 22(5):524-531, May 1999;
incorporated herein by reference).
Demineralized bone matrix was prepared from long bones. The
diaphyseal region was cleaned of any adhering soft tissue and then
ground in a mill. Ground material was sieved to yield a powder with
particles approximately 100 .mu.m to 500 .mu.m in diameter. The
particulate bone was demineralized to less than about 1% (by
weight) residual calcium using a solution of Triton X-100 (Sigma
Chemical Company, St Louis, Mo.) and 0.6N HCl at room temperature
followed by a solution of fresh 0.6N HCl. The powder material was
rinsed with deionized water until the pH was greater than 4.0. It
then was soaked in 70% ethanol and freeze-dried to less than 5%
residual moisture.
Example 3
Formulating Preferred Inventive DBM Compositions
The carrier was prepared by mixing approximately 6.5% (w/w) of the
modified starch, B980, with approximately 30% (w/w) maltodextrin
(M180) and approximately 63.5% (w/w) sterile, deionized water. The
mixture was heated to 70 .degree. C. to pre-gelatinize. The
pre-gelatinized mixture was then transferred into a steam autoclave
and sterilized/gelatinized at 124 .degree. C. for 2 hours. The
resulting mixture then had a consistency of pudding. The cooled
carrier mixture was then combined with DBM (from Example 2) and
water, in a ratio of approximately 27:14:9, respectively. The
stabilized DBM was then implanted into athymic rats to assess
osteoinductivity.
Alternative embodiments: Other components such as glycerol were
added as a solution (approximately 20% w/v) in water instead of
water at the time of pre-gelatinization or during the final
composition mixing and were found to have acceptable handling
characteristics.
Example 4
Stabilized DBM
The table below describes the preparation of a variety of inventive
DBM compositions with different stabilizers. All preparations are
prepared aseptically, and all preparations may be used with DBM
particles, fibers, or solid formed matrices.
1 Class of Stabilizer Stabilizer DBM form Method Diffusion Barrier
Resorbable polymers in Approximately 150-1000 Pre-swollen particles
are powdered form: micron particles lyophilized and mixed with
polymer Tyrosine poly arylate then pre-swollen with 100% powder.
The mixture is Tyrosine polycarbonate glycerol, excess removed by
Melt cast at 60-115 .degree. C. polyorthoester filtration Following
cooling the polymer DBM monolith is pulverized Phosphatidyl choline
Approximately 150-1000 One or more of the
Phosphatidyl-ethanolami-ne micron particles lyophillized indicated
lipids are blended Squalene with the DBM to prepare a Starch
phosphatidyl choline paste containing about 30-80% DBM Masking
Agent Suspend in standard buffer Mix lyophilized DBM with Apply DBM
particles as Lectins system for the specific protein protein
solution to prepare a thick usual or mix with standard Antibodies
or PBS, or 1 mMHCl to a slurry (.about.0.33 gm/mL). Re-DBM carrier
(e.g., Human Anti-concentration ranging from lyophilize. glycerol,
starch, pluronic) noggin about 1 ng/ml to about 10 prior to
application. Human Anti-mg/ml BMP Factor binding proteins Noggin
Chordin TGBP Enzyme Inhibitors TIMPs Soybean trypsin Competitive
Substrates Poly-lys-arg Di-mannose Poly-mannose Poly-L-lysine
Example 5
In Vitro Assessment of Protective Agents
Samples of DBM with carrier with, and without stabilizing agents
(or various concentrations and/or formulations of stabilizing
agents) are prepared and incubated with serum or individual enzymes
(e.g., papain) in pH 7.4 PBS buffer and incubated at 37 .degree. C.
for 0.5, 1, 2, 4, 8, and 24 hours Samples are then extracted to
determine the concentrations of growth factors and other matrix
proteins as outlined in Ueland et. al. ("Increased cortical bone
content of insulin-like growth factors in acromegalic patients" J
Clin Endocrinol Metab 1999 January; 84(1):123-7; incorporated
herein by reference). Samples are prepared for native and
denaturing SDS gel electrophoresis followed by Western blot
analysis or Western Ligand blotting as described in Ueland et al.
(1999) and incorporated herein by reference (Ueland et al
"Increased cortical bone content of insulin-like growth factors in
acromegalic patients" J Clin Endocrinol Metab 1999 January;
84(1):123-7; and Walker, J. M. (Ed) The Protein Protocols Handbook,
Second Edition 2002, Humana Press Totowa, N.J.; each of which is
incorporated herein by reference).
Samples containing stabilizing agents demonstrating less
degradation of growth factors or other proteins than samples
without stabilizing agents are then tested for osteoinductivity at
7, 14, 21, and 28 days in the athymic rat assay. Extract samples
can also be tested rapidly for biological activity in a tissue
culture assay as described in Zhang et al. (1997).
Example 6
Determining Time Course for Induction of Bone Growth by
Intermuscular Implant
This Example characterizes the time course of induction of bone
growth in an intermuscular site using the inventive materials, as
compared with DBM base powder (as in Example 1), at time points of
7, 14, 28, and 35 days. This Example is similar to the rat model
for assessing osteoinduction of DBM found in Edwards et al
"Osteoinduction of Human Demineralized Bone: Characterization in a
Rat Model" Clinical Orthopaedics 357:219-228, December 1998;
incorporated herein by reference.
The study was conducted in athymic (nude) rats in order to minimize
the potential for a cross-species incompatibility response to human
tissue implants. The hind-limb intermuscular site was used for the
initial determination of heterotopic bone induction properties
because the site does not naturally contain bone.
Female homozygous mu/mu rats in the 50-75 g range were obtained
from Harlan (Indianapolis, Ind.). The rats were housed for one week
for acclimatization purposes prior to surgery. Sterile
microisolator cages were used throughout the investigation, with
sterile water and rodent diet provided ad libitum.
Implant Placement: A single intermuscular (IM) site was utilized in
each hind limb of 30 rats. To provide a common positive control
over all animals, a single 40 mg sample of rat DBM powder was
placed intramuscularly within the left pectoralis (LP) muscle of
each rat. Animals were allowed normal activities following surgical
procedures.
Implant Materials: DBM and test materials were kept at room
temperature. Eight 145 mg samples of Test and eight 40-mg samples
of DBM powder were tested for implantation times of 7, 14, and 28
days. Six samples of each were tested at 35 days. The 40 mg samples
of DBM powder were rehydrated with 100 .mu.l of sterile
ALLOPREP.TM. (Ostetotech, Eatontown, N.J.). Each of the samples was
packed into a 1 ml blunt cut syringe. Implantation was randomized
so that a single animal did not receive two of the same
implants.
Anesthesia: The rats were anesthetized with a mixture of ketamine
(200 mg), xylazine (400 mg), and physiological saline (10 ml). The
dosage was 3.5 ml/kg body weight administered
intraperitoneally.
Procedure: Aseptic surgical procedures were carried out in a
laminar airflow hood. A 1-cm skin incision was made on each upper
hind limb using a lateral approach, and the skin was separated from
the muscle by blunt dissection. A superficial incision aligned with
the muscle plane was made to allow for insertion of the tips of the
scissors. Blunt dissection was performed from this line deep into
the muscle to create a pocket to hold the implanted material. A
single suture was inserted to close the muscle pocket, and the skin
was closed with metal clips.
Implantation of specimens in the left pectoralis muscles involved
making a 1-cm skin incision over the chest, blunt dissection of the
muscle to create a pocket, and positioning of the rat DBM powder
using a blunt syringe. A single suture was inserted to close the
muscle pocket, and the skin is closed with metal clips.
Rats were euthanized with CO.sub.2 following the designated
implantation time. Implant materials were located by palpitation,
retrieved by blunt dissection, and cleaned of the surrounding
tissue by careful trimming. An observer blinded to implant type
performed a macroscopic evaluation of the implant material. Color,
vascularity, hardness, and integrity were scored according to the
scheme outlined in the Table below. (The highest score for the most
robust response would be a 4 while a specimen showing little or no
osteoinductive potential would score a 0.) Experience with this
model has shown a high correlation between visual observations and
histological observations of implant performance only at the
extremes of both ends of the scale.
2 Macroscopic Observation Scoring Guidelines Color: .sup. White
(W).sup. .sup. Grey (G) Red (R) Vascularity: None (N) Some (S) sup.
Robust (R).sup. Hardness: Mushy (M).sup. .sup. Firm (F) Hard
(H).sup. Integrity: Diffuse (D).sup. Flat (F) Nodule (N) Score: 0
0.5 1
Histology: Retrieved materials were fixed in Neutral buffered
formalin. After fixation in formalin, samples were decalcified in
10% formic acid, dehydrated in graded alcohols, embedded in JB-4
(glycol methacrylate, Polysciences, Inc., Warrington, Pa.) and
sectioned, Five-micron sections were stained with toluidine blue
and evaluated by light microscopy.
The explants were histologically evaluated using a semiquantitative
method. Briefly, a numerical score based on a five-point scale was
assigned to each section of nodule: 4=more then 75% involved in new
bone formation; 3=51-75% involved in new bone formation; 2=26-50%
involved in new bone formation; 1=1-25% of the explant involved in
new bone formation; and 0=no evidence for the process of
endochondral bone formation including the presence of cartilage or
chondrocytes, active osteoblasts, osteoid, newly formed and
mineralized bone, and/or marrow and associated fat cells.
3 Scoring of Histological Sections Score New Bone Formation 0 No
new bone formation 1 <25% new bone formation 2 26-50% new bone
formation 3 51-75% new bone formation 4 >75% new bone
formation
Following histological analysis, average scores were calculated for
each material type. Based on pervious experience with this animal
model, each group was assigned an assessment of osteoinductive
potential based on the average histological score.
Results: This protocol was followed with the test material, a DBM
with a starch stabilizer as described in example 3, as compared
with control GPS1-2 base DBM powder. At the 7-day timepoint, DBM
with a starch stabilizer and GPS1-2 powder achieved the same level
of induction, with a histologic score of 0.9.+-.0.4 and 1.0.+-.0,
respectively. All samples were hypercellular with a few
chondrocytes present. At the 14-day timepoint, the DBM with a
starch stabilizer achieved a greater level of induction than the
GPS1-2 powder, with a histologic score of 3.6.+-.0.5 and 2.9.+-.1.0
respectively. Clusters of chondrocytes were present in all of the
DBM with a starch stabilizer samples. At this time point, half of
the powder samples also had clusters of chondrocytes, or scattered
cells. At the 28-day point, few chondrocytes were present in either
the DBM with a starch stabilizer or the GPS1-2 powder. Most samples
exhibited mature bone by this stage. Some tissue infiltration was
noted in three of the DBM with a starch stabilizer samples and two
of the powder samples. The histologic score for the DBM with a
starch stabilizer samples and two of the powder samples. The
histologic score for the DBM with a starch stabilizer remained
constant after the 14 days, whereas the histologic score for the
powder improved from 2.9.+-.1.0 to 3.9.+-.0.4 between days 14 and
35 days, without significant change noted for those samples at the
35-day time point
4 Mean Histologic Scores Product 7-Day 14-Day 28-Day 35-Day DBM
(GPS1-2) with a 0.9.+-.0.4 3.6.+-.0.5 3.6.+-.0.5 3.5.+-.0.8 starch
stabilizer DBM (GPS1-2) powder 1.0.+-.0 2.9.+-.1.0 3.9.+-.0.4
3.7.+-.0.5 (Control)
Conclusions: The results of this study indicated that the rate of
induction for the DBM with a starch stabilizer increased to the
14-day timepoint and remained elevated through the end of the time
course. The GPS1-2 powder exhibited induction at a slower rate at
the 14-day time point but was the same as the DBM with a starch
stabilizer samples by 28 days. At this point, the osteoinductive
potential for both products was nearly the same with only a
difference of 0.3 in mean histologic scores and remained the same
at the 35-day time point. The DBM with a starch stabilizer sample
showed a faster rate of bone formation compared to the powder
control. The qualitative evaluation of increased number of
chondrocytes present was indicative of increased bone formation in
the DBM with a starch stabilizer samples.
Example 7
Evaluating Efficacy of Inventive Compositions in Healing Bone
Defects
Background Information: Morselized autogenous cancellous bone (ABG)
has long been considered the "gold standard" for osteoinduction
when a bone graft is required in an orthopedic clinical situation.
Unfortunately, the amount of ABG available is limited, and there is
at least a 5% surgical morbidity associated with the harvesting
procedure. Demineralized bone matrix (DBM) has been shown to have
equal to superior healing potential to ABG. One of the major
disadvantages to demineralized bone matrix is that it often does
not hold the three dimensional space of the defect. Thus, invasion
of the defect site occurs from the surrounding muscle tissue. The
test article, DBM with a starch stabilizer, offers a semi-solid
texture so that the three dimensional space was maintained.
The rabbit ulna defect model has been modified and used in numerous
projects to test the efficacy of osteoinductive and osteoconductive
growth factors and matrices as substitute to autogenous bone graft.
The aim of this study was to evaluate the bone inducing capacity of
the new DBM formulation grafting material in comparison to previous
formulations and ABG.
Materials and Methods:
Study Design Summary:
A. Rabbit bilateral 2-cm ulnar defects.
Treatment groups:
1. DBM+starch
2. Starch Carrier alone
3. Autograft (historical data used for comparison)
Surgical Procedure: Six months old male New Zealand white rabbits
were used. A 2.0 centimeter non-uniting defect was surgically
created in the bilateral ulnae of all rabbits. After complete
periostectomy, thorough defect wash, and partial diaphyseal wash,
grafting was implanted (according to test groups) via open surgical
technique into each defect. The wound was closed primarily in
layers. The test groups are listed in the table below. When
anesthesia was achieved, both forelimbs were shaved and prepared
with the rabbit supine (limbs up) position. Longitudinal incisions
(3-4 cm) were made over both ulnae and the diaphysis (midshaft)
portion of the ulna was exposed. The distal osteotomy was made 1 cm
from the ulnocarpal (wrist) joint and the proximal osteotomy made
3.0 cm from the ulnocarpal joint, to create a 2 cm defect. The
osteotomies were created with a high speed burr. The resultant
loose block of diaphyseal bone was excised with its periosteum
intact. Due to the very adherent interosseous membrane of the
rabbit forelimb, internal fixation was not required. After
irrigation with sterile saline to remove blood, bone, and marrow
remnants, the implant material was placed in the defect. The deep
fascial layer was closed as an envelope around the defect with 3-0
chromic suture. The skin was closed with interrupted nylon suture.
A post-operative dressing/splint was applied and removed on the
fourth post-operative day.
Radiographs: Antero-posterior radiographs were obtained immediately
post-operatively and additional radiographs were taken at 3, 6, 9,
and 12 weeks. High resolution (Faxitron) radiographs were taken of
both limbs after excision and cleaned of soft tissue at either 6 or
12 weeks. Three blinded observers assess each time point for bone
formation and remodeling.
Results: In vivo radiographs at 3 weeks indicated bone formation
was evident in the starch-based formulation (FIG. 1). At 6 weeks,
trabeculation was observed and almost complete bridging of the
critical-sized defect with the starch-based formulation (FIG.
2).
Conclusion: The starch-based formulation appeared to improve the
rate at which bone formation developed.
Example 8
The Following Table Summarizes the Results of Biocompatibility and
Safety Studies for the Starch-Based Diffusion Barrier DBM
Formulation of Example 3
All studies listed in the table below, with the exception of study
#12, were performed by NAMSA (North American Science Associates,
Inc.)--ISO 9001 certified and fully accredited by the Association
for Assessment and Accreditation of Laboratory Animal Care
International (AAALAC). FDA guidelines were followed and NAMSA is
registered with the USDA. All samples submitted to NAMSA were
tested according to laboratory quality guidelines necessary to
assure valid data.
5 Starch Diffusion Barrier DBM Formulation Testing MATERIAL
ABSORBED WITHIN 30 DAYS PASS/FAIL--No. TEST NAME TEST MATERIAL
MODEL METHOD COMMENTS 1 Cytotoxicity Saline Extraction--In vitro
assay--48 hour read Pass--Non-Carrier L-929 mouse cytotoxic
(M180/B980) fibroblasts (4 g:20 ml extract) 2 Hemolysis Saline
Extraction--In vitro assay--Pass--Non Carrier rabbit blood
hemolytic (M180/B980) (4 g:20 ml extract) 3 Pyrogen Saline
Extraction--Intravenous Repeated Pass--Non-Carrier
injection--Rabbit measures 1-3 pyrogenic (M180/B980) hours post (4
g:20 ml extract) injection 4 Genotoxicity a. Saline Extraction--In
vitro--Ames Measurement a. Pass--Non-Carrier (M180/B980) Assay of
revertants mutagenic (4 g:20 ml extract) b. Pass--Non b. DMSO
Extraction--mutagenic Carrier (M180/B980) (4 g:20 ml extract) 5
Acute a. Saline Extraction-Saline extract--4, 24, 48, 72 a.
Pass--Non Systemic Carrier (M180/B980) IV injection--hour reads
toxic Toxicity (4 g:20 ml extract) mice b. Pass--Non b. Cottonseed
oil Cottonseed oil toxic Extraction-Carrier extraction --IP
(M180/B980) injection--mice (4 g:20 ml extract) 6 Sensitization a.
Saline Extraction-Maximization Induction I Induction I: Carrier
(M180/B980) Assay--Guinea (zero time) no (4 g:20 ml extract) pigs
Induction II (6 abnormalities b. Cottonseed oil days) detected
Extraction-Challenge (13 Induction I: Carrier (M180/B980) days post
no (4 g:20 ml extract) induction II)--abnormalities Topical
Application 24, 48, 72 hour detected reads Challenge: No evidence
of causing delayed dermal contact sensitization 7 Intracutaneous a.
Saline Extraction-0.2 ml subcutaneous 24, 48, 72 hour a. Pass--Non
Reactivity Carrier (M180/B980) injection @ five reads irritant (4
g:20 ml extract) separate sites of b. Pass--Non-b. Cottonseed oil
each of 3 rabbits irritant Extraction-Carrier (M180/B980) (4 g:20
ml extract) 8 Muscle a. Final product 6 .times. 2 ml portions 1, 2,
4 and 24 Complies with Implantation (rabbit specific) over
dorsolumbar hour reads clearance of <Study (Pilot) b. Carrier
(M180/B980) region 30 days Clearance in <1 week 9 Muscle a.
Final product Rabbit, Surgical 3 and 7 days a.
Pass--non-Implantation (rabbit specific) Method reads irritant
Study--Histo-b. Carrier (M180/B980) 0.2 ml implanted Irritation and
b. Pass--non-pathology c. Rabbit DBM toxicity irritant powder alone
evaluation a. Pass--non-irritant 10 Implantation--Final Dog; 3 time
points: Clearance<2 Clearance Formulation--Intramuscular 2, 4, 6
weeks. weeks Study Dog specific Implantation To Subsequent
Absorption; Site reads subject Adjacent to Ribs; to 30 cc volume.
bioresorption N=3 profile 11 Systemic a. Final product Rabbit
Sacrifice time Pass Toxicity--(rabbit N=4 for each points at 7, 14,
Histopathology--Intramuscular specific) dose@ each 28, 60 and 90 No
evidence Implantation b. Rabbit DBM time point days subject to of
systemic powder alone N=3 DBM absorption toxicity Using Direct
powder profile. Blood No evidence of Contact controls@ and urine at
72 carrier at 7 Implantation each time point hours. days Low (high
clinical Surgical Blood and Termination of dose, 1.times. @
implantation urine analysis study at 60 3.8 g/rabbit @ along
vertebral with histology days--No approx 1 g/kg) and column and
(liver and changes in High dose (5.times. along ribs for kidney);
hematology or high clinical dose) high dose Evaluation for clinical
implantation gross chemistry anatomical values; lesions No evidence
of systemic toxicity; Evidence of ectopic bone formation 12 Femoral
Final Formulation Rat 12 Weeks; Radiographic Defect Histology and
evidence of X-rays bone formation
Biocompatibility of DBM with a starch stabilizer. Clearance studies
confirmed the removal of DBM with a starch stabilizer carrier from
the implant site in less than 30 days, classifying it as a class B
tissue/bone implant category for ISO 10993 biocompatibility
studies. Four evaluation tests for consideration for Class B
tissue/bone implant category are listed in the ISO guidelines. They
are: cytotoxicity, sensitization, implantation and genotoxicity.
Acute systemic toxicity may also apply in specific cases. In
addition to the suggested four tests, a total of nine additional
safety, biocompatibility, and efficacy studies were performed
(including Example 7). These studies are summarized in the table
above.
Local Reactions
A. Acute intracutaneous injection, and acute muscle implantation
studies were performed. The intracutaneous studies involved both
saline and cottonseed oil extracts of a starch stabilizer. DBM with
a starch stabilizer prepared with Rabbit DBM was used for muscle
implantation. DBM with a starch stabilizer produced minimal
irritation in both studies, being deemed a non-irritant when
compared to the positive control in the muscle implantation and
having a primary index characterization of negligible when
administered as an intracutaneous extract. Intramuscularly applied
DBM without starch carrier, was found to be a moderate
irritant.
B. Cytotoxicity and Genotoxicity. Extracts of a starch stabilizer
demonstrated no ability to induce cell lysis or bacterial
mutagenicity. The cell lysis study employed a saline extract of DBM
with a starch stabilizer. The genotoxicity studies utilized both
saline and DMSO extracts tested on two bacterial species: S.
typhimurium and E. coli.
C. Hemolysis and Pyrogenicity. Saline extracts of a starch
stabilizer were deemed to be non-pyrogenic and non-hemolytic. Body
temperatures in rabbits injected with saline extracts of DBM with a
starch stabilizer gave no indication of pyrogenicity, and the
extract produced a hemolytic index of 0 when added to
anticoagulated pooled rabbit blood.
D. Sensitization. Extracts of the carrier showed no evidence of
delayed dermal contact sensitization. This study employed a saline
and cottonseed oil extracts of the carrier. Guinea pigs received an
intradermal injection of the extracts and, following a recovery
period, were subsequently challenged with a patch of the extract
material.
E. DBM with a starch stabilizer Systemic Safety/Tox. No evidence of
toxicity was observed in studies in which DBM with a starch
stabilizer (rabbit DBM) was implanted intramuscularly (high dose
also had subcutaneous, see below) in the paravertebral muscle, and
animals monitored for 60 days. In these studies, rabbits were
implanted with either approximately 3.5 cc (low dose) or 17.5 cc
(high dose) of DBM with a starch stabilizer (.about.1.1 gm/cc). The
doses on a gm per kilogram basis (.about.1.3 gm/kg; .about.6.41
gm/kg) are approximately equivalent to 5.6.times. and 28.times.
average human implantation dose (15 cc/70 kg or 0.23 gm/kg)
respectively. In the case of the high dose, due to space
limitations in the paravertebral implant site, only 3.5 cc of DBM
with a starch stabilizer were implanted paravertebrally and the
remaining 14 cc were implanted subcutaneously in the dorsal
thorax.
Necropsy results for the test animals failed to show any treatment
effect. Blood chemistries and urinalysis values all fell within the
normal range, with the exception of serum alkaline phosphatase
which was expected to increase due to the induction of ectopic bone
formation in response to DBM with a starch stabilizer.
Example 9
Assaying Osteoinductivity of Test Materials
Objective: The goal of this Example is to assess the
characteristics of various potential Protective Agents, and
particularly to identify those with no negative impact on
osteoinductivity. Preferably, the Protective Agents are easy to
handle, irrigatable, non-toxic, degradable, and moldable (preferred
consistency resembles plumber's putty).
Methods and Materials: This study is conducted using an athymic
(nude) rat model. Preferably, a single DBM preparation is utilized
in all formulations. Potential Protective Agent materials are
sterilized by irradiation. Various Test Compositions, and control
DBM, are implanted into animals, 6-8 sites per material. Each
animal received bilateral intramuscular implantations into the
hindlimbs Each Test Composition contains 40 mg DBM per bone site.
The volume can vary depending on the nature of the carrier.
Results: This protocol was applied to four different Test
Compositions, plus control DBM. The Test Compositions were
implanted into 30 animals; DBM was implanted into 8 individual
animals. Protective Agents were sterilized by autoclaving. The
following Protective Agent Solutions were prepared:
6 G 8% Starch, 28.5% Maltrin 180: 2.8 g Autoclave for 40 minutes
Starch+22.2 g DI water before addition of 10 g Maltrin M180H 8%
Starch, 12.7% glycerol, 28.5% Maltrin Autoclave for 40 minutes 180:
2.8 g Starch+22.2 g 20% glycerol before addition of 10 g solution
Maltrin M180
The following Test Composition formulations were prepared:
7 Implant Preparation Name Composition Recipe E 8% Starch (n=8) 0.4
g of DBM powder mixed with 0.80 g of solution E. Mix thoroughly. F
8% Starch, 18.4% glycerol 0.4 g of DBM powder mixed with (n=8) 0.80
g of solution F. Mix thoroughly. G 8% Starch, 28.5% Maltrin 0.4 g
of DBM powder mixed with 180 (n=8) 0.73 g of solution G. Mix
thoroughly. H 8% Starch, 12.7% glycerol, 0.4 g of DBM powder mixed
with 28.5% Maltrin 180 (n=8) 0.73 g of solution H. Mix thoroughly
Control Human Powder (HDBM)
In addition, as a positive control in every animal, RDBM was placed
in the left pectoralis.
Results:
8 Implant Material Mean SD HDBM--Control Human Pool 2.9 1.0
KF-135-040501-10 Sample E 3.4 0.9 Sample F 3.8 0.5 Sample G 3.6 0.8
Sample H 3.3 1.2
Conclusion: No Test Composition had a negative impact on
osteoinductivity.
Example 10
Osteoinduction in a Rabbit Model
Introduction and methods: Fifty-five male New Zealand White rabbits
were assigned to three treatment groups. Test article was prepared
as described in Example 3. Those animals assigned to the Low Dose
treatment group (n=20) received 3.5 ml of the test article in the
right paravertebral muscle following a protocol specified
procedure. Animals assigned to the High Dose treatment group (n=20)
received 3.5 ml of the test article in the right paravertebral
muscle and 7.0 ml of the test article in the subcutaneous tissue of
each side of the dorsal thoracic area. The animals assigned to the
Control treatment group (n=15) were implanted with 3.5 ml of
control article (rehydrated DBM powder) in the right paravertebral
muscle. At 7, 14, and 28 days post-implantation, four animals from
the Low and High Dose treatment groups and three animals from the
Control groups were humanely sacrificed. At 60 days
post-implantation, the remaining animals were sacrificed (eight
from the Low and High Dose test groups and six from the Control
treatment group). The implant sites were collected from each rabbit
and fixed in 10% neutral buffered formalin (NBF). The test and
control implant sites from the 60 days post-implantation study
interval were placed in decalcification solutions for 3 days after
adequate formalin fixation. All tissue samples were processed using
standard histological techniques, sectioned at 5 .mu.m, and stained
with hematoxylin and eosin.
Results: Osteoinduction was noted in the subcutaneous and
intramuscular implant sites for the test article and in the
intramuscular site for the control DBM (no subcutaneous
implantation at 28 days post-implantation). New bone was
characterized histologically by being slightly more eosinophilic
than the demineralized bone components of the test and control
articles. The new trabeculae were lined by plump (active)
osteoblasts, osteogenic precursors, osteoid, and poorly mineralized
osteoid. In many cases there were osteocytes present and some
evidence of osteonal remodeling. In some cases cartilage was
present. At 60 days post-implantation, the new bone was similarly
characterized, but associated with increased thickness, remodeling,
and frequently loose fibrovascular stroma (morphologically the same
as observed in bone marrow) containing hematopoietic tissue was
observed between the trabeculae. Subjectively, the test article had
a greater degree of bone formation in the muscle implant sites than
the control article. The amount of cartilage present varied between
the implant sites. This variation was most likely due to
differences in the microenvironment for those individual implants.
The precursor cells involved in new bone formation are
pluripotential and under certain microenvironmental conditions will
form fibrous tissue, cartilage, or bone. The cartilage within the
implant sites undergoes endochondral ossification and becomes bone.
Any differences in the tissue response, bone formation, or
cartilage formation between the test article implanted within the
subcutaneous tissue and that implanted in muscle were due to
anatomical and microenvironmental differences between the two
tissues. Bone formation was noted for both the test and control
article implant sites 28 days post-implantation. The amount and
maturity of the bone (as evident by the amount of remodeling and
the presence of loose fibrovascular stroma and hematopoietic
tissue) was greatly increased at 60 days for the test article.
9 Presence of New Bone and Cartilage by Treatment Group and Time
Post-Implantation 7 Days Post-Implantation 14 Days Post-28 Days
Post-60 Days Post-Treatment Group Bone/Cartilage Implantation
Implantation Implantation High Dose 0/0 (n=4) 0/0 (n=4) 2.0/1.5
(n=4) 3.5/0.0 (n=8) Muscle Subcutaneous 0/0 (n=8) 0/0 (n=8) 1.4/1.5
(n=8) 2.7/0.9 (n=15) Low Dose 0/0 (n=4) 0/0 (n=4) 2.3/0.8 (n=4)
4.0/0.4 (n=5) Muscle Control 0/0 (n=4) 0/0 (n=4) 0.7/0.7 (n=4)
2.5/0.2 (n=6) Muscle
The ratings in the table above were based on a 0-4 scale with 0
being 0% of implant area occupied by new (viable) bone/cartilage; 1
being 1-25% of implant area occupied by new (viable)
bone/cartilage; 2 being 26-50% of implant area occupied by new
(viable) bone/cartilage; 3 being 51-75% of implant area occupied by
new (viable) bone/cartilage; and 4 being 76-100% of implant area
occupied by new (viable) bone/cartilage.
Example 11
Terminal Sterilization
This example describes a terminal sterilization method which
minimizes osteoinductivity loss in the inventive preparations.
The inventive DBM preparations are produced in a clean room
environment from human tissue. The finished implants are placed in
individual tray packages.
Each tray is placed in an Audionvac sealing apparatus (Audion
Electro B. V., Weesp-Holland) which is supplied with a cylinder
consisting of 50/50 hydrogen/argon gas. Before the tray packages
are sealed, they are evacuated and backfilled with the gas mixture
twice. Following sealing, the gas mixture remains in each tray
package.
The packaged implants are then sealed packages and then treated
with 15 KGy gamma radiation from a cobalt 60 source to reduce the
bioburden of the implants to the desired level.
Example 12
Comparing Osteoinductivity of DBM Preparations to BMP and/or Other
Growth Factors
In the series of studies presented here, hybrid recombinant human
BMPs (hybrid rhBMPs, hrhBMPs) were prepared possessing stronger
heparin-binding epitopes at the N-termini compared with the wild
type BMP. The heparin-binding site enhances binding to the ECM
increasing local residence time of the BMP so that the potential
for interaction with the appropriate cells in vivo is maximized
(Kubler et al. "EHBMP-2. Initial BMP analog with osteoinductive
properties" Mund Kiefer Gesichtschir. 3 Suppl 1:S134-9, 1999;
incorporated herein by reference).
The aim of the studies was to compare the osteoinductive potential
of hybrid rhBMP-2.times. (hrhBMP-2.times.) with wild-type BMP-2
(rhBMP-2) to determine whether a synergistic potential existed when
hrhBMP-2.times. was combined with a demineralized bone matrix or a
devitalized (inactivated bone matrix).
Methods
To assess the osteoinductive activity of the hybrid rhBMP-2.times.,
1, 5, and 10 .mu.g hrhBMP-2.times. were placed onto 200 mg
osteoinductive human demineralized bone fiber (DBF) matrix and
implanted heterotopically in athymic rats for 21 days (n=6 per
group). The DBF matrix was prepared so that the osteoinductive
potential was approximately 50% of that usually seen so that
differences between treated and untreated DBM were evident.
Controls consisted of osteoinductive human DBF matrix alone,
inactivated human DBF matrix alone ("devitalized", GuHCl extracted)
and inactivated human DBF combined with 1, 5, and 10 .mu.g
hrhBMP-2.times., active and inactivated matrix with 5 .mu.g
wild-type BMP-2. All samples were measured histologically using a
5-point scoring system (score 4=>75% of the cross-sectional area
of the implant with evidence of bone formation, 3=51-75%, 2=26-50%,
1=1-25%, 0=no bone formation) (Edwards J T, Diegmann M H,
Scarborough N L. Osteoinduction of human demineralized bone:
characterization in a rat model. Clin. Orthop. 357:219-28, 1998;
incorporated herein by reference).
Results
Histological scoring as described in the methods section was
inadequate for scoring most of the samples that contained a
morphogen. The devitalized sample alone (inactive DBF matrix)
scored 0; devitalized+1 .mu.g hrh-BMP-2.times. scored 0.8.+-.0.4;
DBF matrix scored 2.5.+-.0.8; all other samples scored 4.
To further distinguish the extent of development of the nodules, a
qualitative scoring system was devised to determine the vascularity
of the sample and residual DBF remaining in the sample. The
following scales were used:
Vascularity (bloody marrow): none=0; minor=1; few vessels, small
vessels=2; moderate cellularity and vessel size=3; extensive
cellularity, large vessels=4
Residual DBF: none=0; minor=1; low=2; moderate=3, extensive=4
The active DBF matrix treated with hrhBMP-2.times. produced a more
differentiated nodule with little residual DBM present, extensive
new bone formation and highly developed vasculature which was not
evident in the devitalized group even at the highest concentration
of morphogen. The devitalized carrier can be compared to the
collagen sponge--essentially an inert, 3-dimensional structure to
support bone growth. The wild-type rhBMP-2 produced a well
developed vasculature and marrow however, the residual bone content
was far greater that the active DBF counterpart.
Conclusion:
The results show that the modified hrhBMP-2.times. possessed
stronger osteoinductive properties than its corresponding wild
type. Ossification was accelerated and the induced bone tissue
showed a denser structure. Synergistic results were obtained when
hrhBMP-2.times. was combined with active DBF matrix and not
devitalized DBF. The most likely explanation for these findings is
the longer half-life of the hrhBMPs-2.times. at the implantation
site. The persistence of the growth factor at the site allowed for
longer interaction time with local cells rather than leaching into
the surrounding tissues resulting in ectopic bone formation sites.
An active matrix substantially increased the osteoinductive
properties of the exogenously added growth factor presumably due to
the combined interactions of many growth factors already present in
demineralized bone (Kubler et al. "Allogenic bone and cartilage
morphogenesis. Rat BMP in vivo and in vitro" J. Craniomaxillofac.
Surg. 19(7):283-8, 1991; Kubler et al. "Effect of different factors
on the bone forming properties of recombinant BMPs" Mund Kiefer
Gesichtschir. 4 Suppl 2:S465-9, 2000; each of which is incorporated
herein by reference).
Example 13
Process of Making a Species-Specific Osteoimplant with Defined
Dimensions
Long bones from human Rhesus Monkey, canine, and rabbit were used
to prepare species-specific solid formed implant matrices. Bones
were aseptically cleaned. The cortical bone was processed in the
bone milling apparatus described in U.S. Pat. No. 5,607,269,
incorporated herein by reference, to yield about 65 grams of
elongate bone fibers. The elongate bone fibers were placed in a
reactor and allowed to soak for about 5-10 minutes in 0.6 N HCl
plus 20-2000 ppm nonionic surfactant solution. Following drainage
of the HCl/surfactant, 0.6 N HCl at 15 ml per gram of total bone
was introduced into the reactor along with the elongate bone
fibers. The reaction proceeded for about 40-50 minutes. Following
drainage through a sieve, the resulting demineralized elongate bone
fibers were rinsed three times with sterile, deionized water at 15
ml per gram of total bone, being replaced at 15-minute intervals.
Following drainage of the water, the bone fibers were covered in
alcohol and allowed to soak for at least 30 minutes. The alcohol
was then drained and the bone fibers were rinsed with sterile,
deionized water. The bone fibers were then contacted with a mixture
of about 4.5 ml glycerol per gram of dry bone fibers and about 10.5
ml sterile deionized water per gram of dry bone fibers s for at
least 60 minutes. Excess liquid was drained and the resulting
liquid composition containing approximately 11% (w/v)
demineralized, elongate bone fibers was transferred to a 11
cm.times.11 cm mold containing a lid having a plurality of
protruding indentations (approximately 1.5 cm.times.3.5 cm in width
and length, and 4 mm in depth), the lid was gently placed on the
mold such that the indentations became immersed into the fibers to
exert as little pressure on the composition as possible. The
dimensions of the protrusions can be made specific for the size of
the osteoimplant required for the animal model of interest. The
resulting cut pieces had dimensions of 4.5 cm in length, 2.5 cm in
width and about 8 mm in height (or thickness) with trough
dimensions 3.5 cm in length, 1 cm in width and depth of the of 4
mm. The mold was then placed in an oven at 46 .degree. C. for 4
hours. The composition was then frozen overnight at -70 .degree. C.
and then lyophilized for 48 hours. Following lyophilization, the
mold was disassembled and the sponge-like formed composition was
cut into individual pieces that contained troughs.
The resulting composition was cohesive, flexible, sponge-like with
an obvious continuous three-dimensional structure with visible open
pores, had a defined shape including the indentations made by the
lid protrusions, did not require rehydration before use, but was
rapidly hydratable and retained its shape once wetted with fluids
and freezing was not required for storage.
Example 14
Method for Fabricating a Partially Embedded DBM/Polymer
Composite
The following method is used to produce a demineralized bone matrix
partially embedded in a resorbable polymer. Such partially embedded
DBMs provide an initial level of osteoinductivity from the
non-embedded DBM portion, and then a continuous source of
un-degraded active DBM as the polymer degrades with time. The
method is particularly well suited for embedding DBM in tyrosine
polycarbonate DT (Integra life sciences) and poly (L-lactide-co-D,
L-lactide 70/30) (Boehringer Ingelheim). This device has particular
application in posterior lateral spine fusion, where it can be
placed within the lateral gutter to promote intertransverse process
bone formation. The method can be used to half embed an
appropriately shaped matrix produced by the method described in
Example 10 above, or alternatively, a collection of demineralized
cortical bone fibers, where the fibers, are cut approximately 1
inch in length and arranged in a cylindrical bundle with the long
axes of the fibers substantially parallel with one another can be
partially embedded by this method.
A stainless steel adjustable diameter circular clamp, approximately
1/2 inch in height is used to hold the ground polymer, along with
the lower portion of the demineralized bone. The fiber bundle or
matrix sample is centered in the clamp, leaving space around the
inside periphery of the clamp to receive the ground polymer
material. Heat is then applied to the underside of the clamp until
the polymer has melted. The clamp is then tightened (diameter
reduced) while the polymer is still flowable, forcing the polymer
to flow into the lower part of the fiber bundle. The polymeric
material is then allowed to cool and the clamp removed, embedding
the lower portion of the fibers in the solid polymer.
In preferred embodiments resorbable polymers are employed.
Temperatures are used which melt the polymer to a suitable
viscosity to allow the melted polymer to flow in and around the
demineralized bone. Most often the temperature employed will be
from about 0 to about 15 degrees above the glass transition
temperature of the polymer. Since the biological activity of DBM
may degrade if maintained at temperatures above 60 .degree. C. for
significant periods of times, the preferred polymers will have
glass transition temperatures lower than 100 .degree. C. preferably
lower than 80 .degree. C. and most preferable below 60 .degree. C.
For tyrosine polycarbonate DT a temperature of 115 .degree. C. for
10 minutes is employed. For poly (L-lactide-co-D, L-lactide 70/30)
70 .degree. C. is suitable. This method is also applicable if a
suitable polymer solvent is used instead of heat to facilitate
polymer flow.
Example 15
DBM Preparation Comprising a Mixture of Stabilized DBMs with a
Prolonged Half-Life Diffusion Barrier
Two demineralized bone formulations are prepared:
Demineralized bone preparation #1. DBM is prepared from about
150-1000 micron bone particles demineralized, lyophilized and then
pre-swollen with 100% glycerol, excess glycerol is removed by
filtration. Lactomer 9-1, a caprolactone glycolide & calcium
stearoyl lactylate (Tyco Inc. North Haven, Conn.) is mixed 10:1 by
weight to homogeneity with the DBM. The mixture is melt cast in a
mold at 70 .degree. C. Following cooling, the polymer DBM monolith
is pulverized in a cryomill and sieved to a particle size of about
130-1200 microns.
Demineralized bone preparation #2: A lecithin based DBM preparation
is prepared according to the method of Han et al "Synergistic
Effects of Lecithin and Human DBM on Bone Induction in Nude Rats"
Abstract from the 28.sup.th Annual Meeting of the Society for
Biomaterials (2002) incorporated herein by reference. Briefly,
Pospholipon 90G, (American Lecithin Company) is mixed with
demineralized bone at a weight ration of between 40% lecithin: 60%
DBM to 60% lecithin: 40% DBM
A third starch based demineralized bone is prepared according to
Example 2 with the exception that only one third of the total
demineralized bone was added to the starch carrier. In place of the
remaining two thirds of demineralized bone, is added equal amounts
demineralized bone from preparations #1 & #2 of this Example.
The composition is then mixed to form the implant preparation.
Example 16
Competitive Substrate
Poly-L-lysine may be used as a competitive inhibitor for serine
protease enzymes. This example describes the preparation of
demineralized bone incorporating poly-L-lysine. Poly-L-lysine
(10-300 kD) is prepared in 1 mM HCL in a range of concentrations
from about 1-10 mg/ml. Demineralized bone is prepared. Following
final washing it is mixed with the poly-L-lysine solution in one of
5 concentrations to form a thick slurry (.about.0.33 gm/mL). The
demineralized bone/substrate mixture is lyophilized to dryness. The
demineralized bone thus prepared is used directly or formulated
with a carrier.
Example 17
A Fatty Acid/Starch Diffusion Barrier Matrix
Demineralized bone is prepared as described in example 14 with the
modification that the polymer/DBM preparation is omitted, being
replaced by an equal weight of the lecithin preparation.
Example 18
Osteoinduction of DBM Composition in an Athymic Rat Model
The purpose of this Example is to evaluate the osteoinductive
potential of DBM compositions using a heterotopic osteoinductive
28-day implant model (Edwards et al., Clin. Orthop. Rel. Res.
357:219-228, 1998; Urist, Science 150:893-899, 1965; each of which
is incorporated by reference). The DBM composition includes
cuboidal shaped DBM particles in combination with DBM fibers (See
U.S. Ser. No. 60/159,774, filed Oct. 15, 1999; WO0232348; each of
which is included herein by reference). Chondrocytes are the
predominant cell type in the cube of the DBM following 28-day
implantation. This study extends the implant time to 49 days to
look evidence of continued bone remodeling within the demineralized
cortical cube.
Materials and Methods: Equal volumes of crunch samples weighing
approximately 600 mg were packaged in 2.5 ml blunt tipped syringes.
Eighteen female athymic rats were obtained from Harlan Sprague
Dawley Inc. (Indianapolis, Ind.). Animals weights at the time of
surgery ranged between 186 g and 236 g. 28-day and 49-day implants
were evaluated.
The implant sites were assessed histologically. The fiber component
was scored independently of the cubes and was assigned a numerical
score based on a 5 point semiquantitative scale based on percent of
fiber area involved in new bone formation. The cube portion was
assigned a score based on the percent of central Haversian systems
involved in new bone formation.
Results: New bone, marrow, and adipocytes were present throughout
the fiber portion of the nodules. Chondrocytes were present within
the central Haversian systems at all time points. At the 28-day
time point, the mean osteoinductive score for the fiber portion was
3.1.+-.0.5 for the fiber portion 89.8.+-.5.8% of the Haversian
canals occupied in the cube portion. Cubes were surrounded by new
bone or marrow and pockets of chondrocytes occurred within and
between cubes.
The mean osteoinductive score at the 49-day time point was
3.5.+-.0.5 for the fiber portion with 98.1.+-.2.4% of the Haversian
canals occupied in the cubes. The notable differences from the
28-day samples included almost complete remodeling of the fiber
portion, large pockets of chondrocytes and areas of new bone within
the cubes and remodeling at the edges of the cube.
Conclusions: The cortical cubes play an important role in the
osteoinductivity of the DBM composition. The cubes are cut from
cortical bone and the central Haversian canals provide a natural
porosity. Cartilage persisting after 28 days coincides with a delay
in bone formation presumably due to the delayed vascular ingrowth.
At 49 days, the cubes showed evidence of remodeling albeit slower
than the fibers. Bone remodeling occurred faster on the external
surfaces compared to internal surfaces. The cubes continue to
provide the important support matrix and osteoinductive signal
required for normal bone formation throughout the healing
response.
Example 19
Establishment of Handling Characteristics for Inventive
Compositions
The following example describes the addition of demineralized bone
to an inventive stabilizing agent and/or diffusion barrier to
produce a formable osteoinductive implant composition. The example
describes the establishment of an appropriate carrier viscosity,
mixing the carrier with DBM, and adjustment of the final handling
properties of the competed composition.
Carrier Viscosity. The inventive starch based compositions were
prepared as described in Example 3, with a variety of starch to
water ratios ranging from about 5% to about 45%. The starch powders
was mixed with water and the mixture was autoclaved to produce a
sterile hydrated starch preparation. The autoclaved starch was then
tested for viscosity. Starch formulations with viscosities within
the range of 5000 to 20000 sCp were used to prepared DBM
compositions.
A Brookfield Viscometer (HB-DV III+) with the appropriate sample
adaptor (SSA27/13RPY s/n RP66162 with spindle #27), supported by
Rheocalc32 software was used to determine the viscosity of the
starch carrier.
Mixing of carrier and DBM. The starch carrier with a viscosity of
approximately 5000 sCpi was mixed with varying quantities of DBM
(from about 10% to about 50% DBM by weight) to produce a
composition with a consistency similar to that of modeling clay or
bread dough. Variations employing lesser amounts of DBM resulted in
a composition with a cohesive yet almost flowable product.
Formulations employing more DBM produced a product with a very
stiff consistency, and formulations with high levels of DBM became
crumbly and fragmented while mixing. These formulations were then
quantitatively assessed for handling as described below.
Assay for composition handling properties. The following method was
used to establish consistency in handling properties of the
inventive compositions. Compositions employing starch-based
carriers with penetration resistance values of 25-120N were
considered acceptable, with values of 30-90N representing a more
preferable range and values between 40-65 N being even more
preferable.
A 1'' diameter.times.9'' long threaded (14 tpi) push rod was
mounted to the actuator of a MTS 858 Bionix Test System fitted with
a 1 kN force transducer. A piece of 1.5'' diameter.times.6'' length
PVC pipe was centered vertically on the force transducer and a
large weigh boat was placed underneath it to catch the extruded
bone formulation. A 1'' I.D..times.0.5'' thick spacer was placed on
top of the PVC tubing and 7.00 g of bone mixture was weighed into a
5 cc syringe and loaded into the tip of the syringe using a clean,
dry 5 cc syringe plunger with the tip removed just below the o-ring
to create a flat surface. The loaded syringe was placed vertically
into the spacer/PVC pipe assembly with the plunger facing up. The
whole assembly (PVC pipe, spacer, and syringe) was centered on the
load cell directly under the push rod. The center of the plunger
was lined up with the center of the push rod. The 1 kN load range
was used for the first test of each new bone formulation. When the
maximum load required to extrude the bone mixture was less than
90N, then the 100N load range was used during subsequent tests to
achieve a higher degree of accuracy. The test sample was preloaded
under load control to 5N, the displacement was zeroed, and the test
was executed. Bone formulations were extruded at a rate of 5 mm/min
to a maximum displacement of 20 mm in compression. The average
maximum force required to extrude each bone formulation was then
determined.
Example 20
Detection of Amylase Sensitivity
This example describes the assessment of amylase resistance for
starch-based stabilizers and diffusion barriers (carriers).
Increasing the amylase resistance of a starch-based carrier
increases the effective residence time of the carrier following
implantation and therefore enhances the stabilizing effect of the
carrier.
Quantification of resistant starches requires the use of pancreatic
a amylase and amyloglucosidase that effectively detect the
breakdown of amylase-resistant starches to glucose.
The breakdown of the starch and starch/lipid compositions of
Examples 3, 9, 15, and 17 as well as new candidate amylase
resistant starches and modified starches, is monitored using the
resistant starch assay kit from Megazyme International Ireland Ltd.
(Amyloglucosidase alpha.-Amylase Method AOAC Method 996.11, AACC
Method 76.13, ICC Standard Method No. 168). Formulations with
slowest breakdown will generally have the longest stabilization
effect in vivo.
Example 21
Starch/Lipid Carrier Compositions
The following compositions were prepared in a similar fashion to
those described in Examples 3, 9, 15, and 17. Carriers were
autoclaved for 20 minutes to sterilize them prior to mixing with
DBM.
Combination #1--Carrier 1 consisted of about 8% Penford Maps 281
and 5% Lecithin with the remainder being water.
Combination #2--Carrier 2 consisted of about 8% Penford Maps 281
and 15% Lecithin with the remainder being water.
Combination #3--Carrier 3 consisted of about 6% GPC B980 and 5%
Lecithin with the remainder being water.
Combination #4--Carrier 4 consisted of about, 6% GPC B980 and 15%
Lecithin with the remainder being water.
Each of the four carrier combinations were mixed with human DBM to
yield a bone content of about 25%. These bone mixtures were then
tested for osteoinductivity as previously described in Example
6.
Other Embodiments
The foregoing has been a description of certain non-limiting
preferred embodiments of the invention. Those of ordinary skill in
the art will appreciate that various changes and modifications to
this description may be made without departing from the spirit or
scope of the present invention, as defined in the following
claims.
* * * * *
References